Engineered proline hydroxylase polypeptides and methods

ABSTRACT

The present disclosure provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

The present application is a Continuation of U.S. patent applicationSer. No. 16/446,866, filed Jun. 20, 2019, now U.S. Pat. No. 10,731,189,which is a Continuation of U.S. patent application Ser. No. 15/357,668,filed Nov. 21, 2016, now U.S. Pat. No. 10,370,688, which is a Divisionalof U.S. patent application Ser. No. 14/399,034, filed Nov. 5, 2014, nowU.S. Pat. No. 9,790,527, which is a national stage application filedunder 35 USC § 371 and claims priority to PCT International ApplicationNo. PCT/US2013/039874, filed May 7, 2013, and U.S. Prov. Appln. Ser. No.61/644,135, filed on May 8, 2012. The present application herebyincorporates each of these applications by reference, in theirentireties and for all purposes.

1. TECHNICAL FIELD

The disclosure relates to biocatalysts for the hydroxylation of chemicalcompounds.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-095WO2_ST25.txt”, a creation date of May 2, 2013, anda size of 433,542 bytes. The Sequence Listing filed via EFS-Web is partof the specification and is incorporated in its entirety by referenceherein.

3. BACKGROUND

Proline derivatives with functional groups on the ring carbons areuseful building blocks for synthesis of pharmaceutical compounds becauseof the constrained conformation of proline. One such derivative,hydroxylated proline, is a starting material for the synthesis ofvarious therapeutic compounds, including carbapenem antibiotics (see,e.g., Altamura et al., 1995, J Med Chem. 38(21):4244-56),angiotensin-converting enzyme inhibitors, protease inhibitors (see,e.g., Chen et al., 2002, J Org Chem. 67(8):2730-3; Chen et al., 2006, JMed Chem. 49(3):995-1005), nucleic acid analogs (Efimov et al., 2006,Nucleic Acids Res. 34(8):2247-2257), isoprenyltransferase inhibitors(O'Connell et al., 2000, Chem Pharm Bull. 48(5):740-742), and druglibrary construction (Vergnon et al., 2004, J Comb Chem. 6(1):91-8;Remuzon P., 1996, Tetrahedron 52:13803-13835). Similarly, hydroxylatedderivatives of a proline homolog, L-pipecolic acid, also known ashomoproline, also serve as building blocks for pharmaceutical compounds.For example, hydroxypipecolic acid is an intermediate in the synthesisof β-lactamase inhibitors (see, e.g., WO2009091856, WO2010126820 andUS20110046102) and TNF-alpha converting enzyme (TACE) inhibitors(Levatic et al., 2002, Bioorg Medicinal Chem Lett. 12(10):1387-1390).

Hydroxyproline can be obtained from natural sources, such as plantmaterials and hydrolyzates of collagen. Hydroxyproline can also bechemically synthesized, such as from starting materials allyl bromideand diethylacetamidomalonic acid (Kyun Lee et al., 1973, Bull. Chem.Soc. Japan, 46:2924), D-glutamic acid (Eguchi et al., 1974, Bull. Chem.Soc. Japan, 47:1704-08), glyoxal and oxaloacetic acid (Ramaswamy et al.,1977, J. Org. Chem. 42(21):3440-3443), and β-alanine (Sinha et al.,2000, Proc. ECSOC-4, The Fourth International Electronic Conference onSynthetic Organic Chemistry, ISBN 3-906980-05-7).

Hydroxypipecolic acid can also be obtained from plants and other naturalsources (see, e.g., Romeo et al., 1983, Phytochemistry 22(7):1615-1617;Fowden, L., 1958, Biochem J. 70(4):629-33; Clark-Lewis and Mortimer,1959, Nature 184(Suppl 16):1234-5). Chemical synthesis ofhydroxypipecolic acid is described in Callens et al., 2010, Bulletin desSociétés Bulletin des Sociétés Chimiques Beiges 91(8):713-723; Adams etal., 1996, Chem. Commun. 3:349-350; Botman et al., 2004, Organic Letters6(26):4941-4944; Cohen et al., 1956, Science 123(3202):842-843; Beyermanet al., 1959, Recueil des Travaux Chimiques des Pays-Bas, 78(9):648-658;Marin et al., 2004, J Org Chem. 69(1):130-41; Kumar et al., 2005, J OrgChem. 70(1):360-3; Liang et al., 2005, J Org Chem. 70(24):10182-5;Kalamkar et al., 2008, J Org Chem. 73(9):3619-22; Chiou et al., 2010, JOrg Chem. 75(5):1748-51; Lemire et al., 2010, J Org Chem. 75(6):2077-80;and Angelique et al., 2000, Tetrahedron Lett. 41(36):7033-7036.

Isolation from natural sources is limited by the availability of rawmaterials, requires purification from a significant amount of backgroundcontaminants, and lacks certain desired diastereomers. Chemicalsynthetic methods can require complex steps, be difficult to scale up toindustrial scale levels, and require additional purification steps dueto formation of multiple hydroxylated products.

Another approach for preparing hydroxylated proline uses prolinehydroxylases, which are 2-oxoglutarate-dependent dioxygenases, utilizing2-oxoglutarate (α-ketoglutarate) and O₂ as co-substrates and ferrous ionas a cofactor (see, e.g., Klein et al., 2011, Adv Synth. Catal.353:1375-1383; U.S. Pat. No. 5,364,775; and Shibasaki et al., 1999, ApplEnviron Microbiol. 65(9):4028-4031). Unlike prolyl hydroxylases thatspecifically recognize peptidyl proline in procollagen and relatedpeptides, proline hydroxylases are capable of converting free proline tohydroxyproline. Several microbial enzymes that produce cis-3-, cis-4- ortrans-4-hydroxyproline are known (see, e.g., U.S. Pat. Nos. 5,962,292;5,963,254; 5,854,040; WO2009139365; and EP2290065) and an enzyme thatproduces trans-3-hydroxyproline have been identified in extracts of thefungus Glarea lozoyensis. Many of the proline hydroxylases are found inbacteria, where they are associated with the biosynthesis of peptideantibiotics. The cis-4-proline hydroxylase enzyme also shows activity inconverting L-pipecolic acid (i.e., (2S)-piperidine-2-carboxylic acid) tocis-5-hydroxypipecolic acid (i.e.,(2S,5S)-5-hydroxypiperidine-2-carboxylic acid; Klein et al. supra). Invitro conversions for preparing 5-hydroxypipecolic acid using theseenzymes have been demonstrated, but isolated proline hydroxylases arefound to denature under reaction conditions and have relatively lowspecific activity, rendering in vitro uses impracticable for commercialapplications (Klein et al., supra). While recombinant whole cellsexpressing cloned proline hydroxylases are better suited for large scaleindustrial processes, the use of whole cells limits variations inreaction conditions, such as high substrate concentrations; restrictsthe types of substrates that can be used with whole cells to those thatare permeable to the cells; and results in undesirable by-products thatmust be separated from the final product. In addition, in vivo systemsmay require defined growth media not optimal or cost effective becausethe use of rich growth media prepared from protein hydrolyzates containfree proline, which can be a competitive inhibitor when substrates otherthan proline are being targeted. Desirable are alternative methods forsynthesizing hydroxylated forms of proline and proline analogs, as wellas other chemical compounds, that can be readily scaled up and result insubstantially pure stereomeric product.

4. SUMMARY

The present disclosure provides engineered proline hydroxylasebiocatalysts, polynucleotides encoding the biocatalysts, methods oftheir preparation, and processes for preparing hydroxylated compoundsusing these engineered biocatalysts. The proline hydroxylases of thepresent disclosure have been engineered to have improved propertiesrelative to the naturally occurring cis-4-proline hydroxylase (SEQ IDNO:2) of Sinorhizobium meliloti, a nitrogen fixing Gram negativebacterium. The improved biocatalyst properties of the engineered prolinehydroxylases include, among others, activity, regioselectivity,substrate tolerance, and stability. The engineered proline hydroxylasesare also found to hydroxylate a variety of substrate compounds,including the selective conversion of (2S)-piperidine-2-carboyxlic acid(i.e., L-pipecolic acid) to (2S,5S)-5-hydroxylpiperidine-2-carboxylicacid (i.e., cis-5-hydroxypipecolic acid). The engineered enzymes withimproved properties have one or more residue differences as compared tothe naturally occurring proline hydroxylase, where the residuedifferences occur at residue positions affecting the foregoing enzymeproperties.

Accordingly, in one aspect, the present disclosure provides engineeredpolypeptides having proline hydroxylase activity, where the polypeptidescomprises an amino acid sequence having at least 80% identity to SEQ IDNO:2 and one or more residue differences as compared to SEQ ID NO:2 atresidue positions selected from: X2; X3; X4; X5; X9; X13; X17; X24; X25;X26; X29; X30; X36; X42; X52; X57; X58; X59; X62; X66; X86; X88; X92;X95; X98; X103; X112; X113; X114; X115; X116; X121; X131; X140; X150;X151; X166; X186; X188; X205; X225; X230; X270; and X271.

In some embodiments, the residue differences at residue positions X2;X3; X4; X5; X9; X13; X17; X24; X25; X26; X29; X30; X36; X42; X52; X57;X58; X59; X62; X66; X86; X88; X92; X95; X98; X103; X112; X113; X114;X115; X116; X121; X131; X140; X150; X151; X166; X186; X188; X205; X225;X230; X270; and X271 can be selected from X2K; X2T; X3S; X4Q; X4L; X4E;X4S; X5I; X5L; X5M; X9I; X13T; X17V, X24R; X24S; X25R; X26R; X26T; X26W;X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G; X62Q; X66Q;X86S; X88R; X92V; X95M; X98F; X98T; X103L; X103Q; X112T; X112V; X113E;X114N; X115E; X115H; X115D; X115G; X1155; X115A; X116L; X121F; X131Y;X131F; X140L; X1505; X151A; X151H; X1515; X166T; X166L; X166Q; X186G;X188G; X205V; X225L; X225Y; X225W; X230V; X270E; X271K; and X271R. Thefollowing detailed description provides guidance on the choices of theresidue differences that can be used to prepare engineered prolinehydroxylases with the desired improved biocatalytic properties.

In some embodiments, the engineered proline hydroxylase polypeptide hasan amino acid sequence comprising at least a combination of featuresselected from: (a) X103L and X166Q; (b) X52P and X255Y; (c) X4E/L/S andX115A; (d) X25R and X58A; (e) X29A and X166T/Q/L; (f) X115H/D/G andX121F; (g) X3S, X103L, and X166Q; (h) X103L, X131Y/F, and X166T/Q/L; (i)X26T, X103L and X166T/Q/L; (j) X25R, X66Q, X92V and X115E; (k) X25R,X66Q, X92V, X103L, X115E, and X166Q; and (l) X3S, X25R, X66Q, X92V,X103L, X115E, and X166Q.

As noted above, the engineered polypeptides having proline hydroxylaseactivity, are also capable of converting substrate compound (2),(2S)-piperidine-2-carboxylic acid to product compound (1),(2S,5S)-5-hydroxylpiperidine, as shown in Scheme 1,

with improved properties as compared to the naturally occurring enzyme.In some embodiments, the engineered polypeptides are capable ofconverting substrate compound (2) to product compound (1) with at least1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, or 10 fold or morethe activity of the naturally occurring enzyme, and with greater than90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more diastereomericexcess of (2S,5R)-5-hydroxypiperidine-2-carboxylic acid.

In some embodiments, the engineered polypeptides having prolinehydroxylase activity also display increased regioselectivity for forming(2S,5S)-5-hydroxypiperidine-2-carboxylic acid in excess of otherregioisomers, for example (2S,3R)-3-hydroxypiperidine-2-carboxylic acid,shown in Scheme 1 as compound (1a). Thus, in some embodiments, theengineered proline hydroxylases are capable of converting substratecompound (2) to product compound (1) in excess of product compound (1a),where the ratio of product compound (1) formed over product compound(1a) is at least 1.5, 2, 3, 4, 5 or 6 or more. In some embodiments, theengineered proline hydroxylases with increased selectivity for formingproduct compound (1) in excess of product compound (1a) comprises anamino acid sequence having one or more of the following features: X103L;X115E; X131Y and X166Q, particularly the combination of features X103Land X166Q.

In some embodiments, the engineered polypeptides having improvedproperties has an amino acid sequence comprising a sequence selectedfrom the group consisting of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, and 228.

In another aspect, the present disclosure provides polynucleotidesencoding the engineered proline hydroxylase polypeptides with improvedproperties. Exemplary polynucleotide sequences are provided in theSequence Listing incorporated by reference herein and include SEQ ID NO:7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, and 227.

In a further aspect, the present disclosure also provides codonoptimized polynucleotides encoding a polypeptide that comprises an aminoacid sequence of the wild-type proline hydroxylase (SEQ ID NO:2). Insome embodiments, the codon optimized polynucleotides have increasedexpression in a bacterial host cell as compared to the naturallyoccurring polynucleotide encoding the proline hydroxylase enzyme. Insome embodiments, the codon optimized polynucleotides can have at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentity to the codon optimized nucleic acid sequence of SEQ ID NO: 1,3, or 5, which encodes the identical polypeptide sequences of SEQ IDNO:2, 4, or 6, respectively. The codon optimized sequence of SEQ IDNO:1, 3, or 5 can enhance expression of the encoded, wild-type prolinehydroxylase by at least 1.2 fold, 1.5 fold or 2 fold or greater ascompared to the naturally occurring polynucleotide sequence.

In another aspect, the polynucleotides of the disclosure can beincorporated into expression vectors and host cells for expression ofthe polynucleotides and the corresponding encoded proline hydroxylasepolypeptides. As such, in some embodiments, the present disclosureprovides methods of preparing the proline hydroxylase polypeptides byculturing a host cell comprising the polynucleotide or expression vectorcapable of expressing a proline hydroxylase of the disclosure underconditions suitable for expression of the engineered polypeptide. Insome embodiments, the method of preparing the proline hydroxylase cancomprise the additional step of isolating the expressed polypeptide.

In some embodiments, the present disclosure also provides methods ofmanufacturing an engineered proline hydroxylase polypeptide, where themethod can comprise: (a) synthesizing a polynucleotide encoding apolypeptide comprising an amino acid sequence selected from SEQ ID NO:8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, and 228, and having one or more residue differences as compared toSEQ ID NO:2 at residue positions selected from: X2; X3; X4; X5; X9; X13;X17; X24; X25; X26; X29; X30; X36; X42; X52; X57; X58; X59; X62; X66;X86; X88; X92; X95; X98; X103; X112; X113; X114; X115; X116; X121; X131;X140; X150; X151; X166; X186; X188; X205; X225; X230; X270; and X271,and (b) expressing the proline hydroxylase polypeptide encoded by thepolynucleotide. As noted above, the residue differences at positions X2;X3; X4; X5; X9; X13; X17; X24; X25; X26; X29; X30; X36; X42; X52; X57;X58; X59; X62; X66; X86; X88; X92; X95; X98; X103; X112; X113; X114;X115; X116; X121; X131; X140; X150; X151; X166; X186; X188; X205; X225;X230; X270; and X271 are selected from X2K; X2T; X3S; X4Q; X4L; X4E;X4S; X5I; X5L; X5M; X9I; X13T; X17V, X24R; X24S; X25R; X26R; X26T; X26W;X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G; X62Q; X66Q;X86S; X88R; X92V; X95M; X98F; X98T; X103L; X103Q; X112T; X112V; X113E;X114N; X115E; X115H; X115D; X115G; X115S; X115A; X116L; X121F; X131Y;X131F; X140L; X150S; X151A; X151H; X151S; X166T; X166L; X166Q; X186G;X188G; X205V; X225L; X225Y; X225W; X230V; X270E; X271K; and X271R. Asfurther provided in the detailed description, additional variations canbe incorporated during the synthesis of the polynucleotide to prepareengineered proline hydroxylases with corresponding differences in theexpressed amino acid sequences.

In another aspect, the engineered proline hydroxylase polypeptides canbe used in a process for preparing various hydroxylated compounds, suchas hydroxylated proline or hydroxylated piperidine-2-carboxylic acids.Accordingly, in some embodiments, the engineered proline hydroxylasepolypeptides can be used in a process for the conversion of a substratecompound of formula (II) to product compound of formula (I), as shownbelow:

wherein

L is selected from the group consisting of a bond, (C₁-C₄)alkylene and(C₂-C₄)alkenylene;

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² and R³ are each independently selected from the group consisting ofhydrogen and optionally substituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and(C₂-C₆)alkynyl;

R⁴ is selected from the group consisting of optionally substituted(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, aryl, heteroaryl,cycloalkyl, heterocycloalkyl; or R⁴ together with one of R¹ or R² is a(C₁-C₅)alkylene or (C₂-C₅)alkenylene and forms a 5- to 8-memberedheterocyclic ring containing the nitrogen atom, wherein the ring isoptionally substituted with 1 to 4 independently selected R⁶ groups;

R⁵ is hydrogen or a bond that forms an epoxide with a carbon atom of L;

each occurrence of R⁶ is independently selected from the groupconsisting of halo, (C₁-C₆)alkyl, and (C₁-C₆)alkyloxy; and

----- represents an optional bond to a carbon atom of L to form a doublebond;

with the provisos that

(i) when R⁴ does not form a ring with one R² or R³, or when R⁴ forms a5-membered heterocyclic ring containing the nitrogen atom with one of R²or R³, then L is a methylene;

(ii) when R⁴ forms a 6-membered heterocyclic ring containing thenitrogen atom with one of R² or R³, then L is a bond or ethylene; and

(iii) when R⁵ is a bond to a carbon atom of L to form an epoxide, thenR⁴ forms the heterocyclic ring containing the nitrogen atom with one ofR² or R³ and L is a (C₁-C₄)alkylene or (C₂-C₄)alkenylene.

In some embodiments, the engineered proline hydroxylase can be used forthe conversion of a ring compound of structural formula (IIa) to thehydroxylated product compound of structural formula (Ia);

wherein

Q is selected from the group consisting of a (C₁-C₅)alkylene and(C₂-C₅)alkenylene;

L is selected from the group consisting of a bond, (C₁-C₄)alkylene and(C₂-C₄)alkenylene;

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² is selected from the group consisting of hydrogen and optionallysubstituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and (C₂-C₆)alkynyl;

R⁵ is hydrogen, or a direct bond to a carbon atom of L to form anepoxide;

each occurrence of R⁶ is selected from the group consisting of halo,(C₁-C₆)alkyl, and (C₁-C₆)alkyloxy;

q is an integer from 0 to 4; and

-   -   represents an optional bond to a carbon atom of L to form a        double bond;

wherein the sum of ring carbon atoms for Q+L is an integer from 2 to 5;

----- with the provisos that

(i) when the sum of ring carbon atoms for Q+L is 2, then L is amethylene; and

(ii) when the sum of ring carbon atoms for Q+L is 3, then L is either abond or ethylene.

In some embodiments, the engineered proline hydroxylase can be used forthe conversion of a substrate compound of structural formula (IV) to thehydroxylated product compound of structural formula (III);

wherein,

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² and R³ are each independently selected from the group consisting ofhydrogen and optionally substituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and(C₂-C₆)alkynyl; and

R⁴ is selected from the group consisting of optionally substitutedalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, andheterocycloalkyl.

In some embodiments, the engineered proline hydroxylase can be used forthe conversion of a substrate compound of structural formula (VI) to thehydroxylated product compound of structural formula (V);

wherein,

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² and R³ are independently selected from the group consisting ofhydrogen and optionally substituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and(C₂-C₆)alkynyl;

each occurrence of R⁶ is independently selected from the groupconsisting of halo, (C₁-C₆)alkyl, and (C₁-C₆)alkyloxy;

R⁷ is selected from the group consisting of hydrogen, halo, andoptionally substituted (C₁-C₆)alkyl and (C₁-C₆)alkyloxy; or R⁷ togetherwith one of R² or R³ forms a 5- to 7-membered heterocyclic ringcontaining the nitrogen atom;

q is an integer from 0 to 4; and

----- represent optional double bonds that form an aryl ring.

The hydroxylation reactions above using the engineered prolinehydroxylases are carried out under suitable reaction conditions inpresence of a co-substrate (e.g., α-ketoglutarate), a divalenttransition metal (e.g., Fe⁺²), and molecular oxygen (i.e., O₂). Thesuitable reaction conditions can comprise ranges of co-substrate,divalent transition metal, molecular oxygen as well as ranges of otherparameters, such as reductant (e.g., ascorbic acid) concentration,detergent concentration, pH, temperature, buffer, solvent system,substrate loading, polypeptide loading, pressure, and reaction time. Insome embodiments, the hydroxylation reaction can be carried out, whereinthe engineered proline hydroxylase is immobilized on a solid support.

In some embodiments, the suitable reaction conditions can comprise thefollowing: (a) substrate loading at about 5 g/L to 30 g/L; (b) about 0.1g/L to 10 g/L of the engineered polypeptide; (c) about 19 g/L (0.13 M)to 57 g/L (0.39 M) of α-ketoglutarate; (d) about 14 g/L (0.08 M) to 63g/L (0.36 M) ascorbic acid; (e) about 1.5 g/L (3.8 mM) to 4.5 g/L (11.5mM) of FeSO₄; (f) a pH of about 6 to 7; (g) temperature of about 20° to40° C.; and (h) reaction time of 2-24 h. In some embodiments, thesuitable reaction conditions comprise forced aeration of the reactionsolution with 02 at a rate of about 3 L/h.

In some embodiments, the suitable reaction conditions can comprise thefollowing: (a) substrate loading at about 10 g/L to 100 g/L; (b) about 1g/L to about 50 g/L of engineered polypeptide; (c) α-ketoglutarate atabout 1 to 2 molar equivalents of substrate compound; (d) ascorbic acidat about 0.25 to 0.75 molar equivalents of substrate compound; (e) about0.5 mM to about 12 mM of FeSO₄; (f) pH of about 6 to 8; (g) temperatureof about 20° to 40° C.; and (h) reaction time of 6 to 120 h. In someembodiments, the suitable reaction conditions comprise forced aerationof the reaction solution with 02 at a rate of about 2 L/h to about 5L/h.

Guidance on the choice of engineered proline hydroxylases, preparationof the biocatalysts, the choice of enzyme substrates, and parameters forcarrying out the processes are further described in the detaileddescription that follow.

5. DETAILED DESCRIPTION

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly indicates otherwise. Thus, for example, reference to “apolypeptide” includes more than one polypeptide.

Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of this disclosure.

The section headings used herein are for organizational purposes onlyand not to be construed as limiting the subject matter described.

5.1 Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine HIS H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleosides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

5.2 Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings:

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Polynucleotide” or “nucleic acid” refers to two or more nucleosidesthat are covalently linked together. The polynucleotide may be whollycomprised ribonucleotides (i.e., an RNA), wholly comprised of 2′deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′deoxyribonucleotides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), itmay include one or more modified and/or synthetic nucleobases, such as,for example, inosine, xanthine, hypoxanthine, etc. Preferably, suchmodified or synthetic nucleobases will be encoding nucleobases.

“Proline hydroxylase” refers to a polypeptide having an enzymaticcapability of converting free proline to hydroxyproline in presence ofco-substrate α-ketoglutarate and dioxygen, as illustrated below:

It is to be understood that proline hydroxylases are not limited to theforegoing reaction with proline, but may hydroxylate other substrates,for example pipecolic acid. Proline hydroxylases as used herein includenaturally occurring (wild-type) proline hydroxylase as well asnon-naturally occurring engineered polypeptides generated by humanmanipulation.

“Co-substrate” of a proline hydroxylase refers to α-ketoglutarate andco-substrate analogs that can replace α-ketoglutarate in hydroxylationof proline and proline substrate analogs. Co-substrate analogs include,by way of example and not limitation, 2-oxoadipate (see, e.g., Majamaaet al., 1985, Biochem. J. 229:127-133).

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” or “non-naturally occurring” when usedwith reference to, e.g., a cell, nucleic acid, or polypeptide, refers toa material, or a material corresponding to the natural or native form ofthe material, that has been modified in a manner that would nototherwise exist in nature, or is identical thereto but produced orderived from synthetic materials and/or by manipulation usingrecombinant techniques. Non-limiting examples include, among others,recombinant cells expressing genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence for optimal alignment of the two sequences. Thepercentage may be calculated by determining the number of positions atwhich the identical nucleic acid base or amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Alternatively, the percentage may becalculated by determining the number of positions at which either theidentical nucleic acid base or amino acid residue occurs in bothsequences or a nucleic acid base or amino acid residue is aligned with agap to yield the number of matched positions, dividing the number ofmatched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Those of skill in the art appreciate that there aremany established algorithms available to align two sequences. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch,1970, J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examplesof algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas, the neighborhood word score threshold (Altschul et al, supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplarydetermination of sequence alignment and % sequence identity can employthe BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredproline hydroxylase, can be aligned to a reference sequence byintroducing gaps to optimize residue matches between the two sequences.In these cases, although the gaps are present, the numbering of theresidue in the given amino acid or polynucleotide sequence is made withrespect to the reference sequence to which it has been aligned.

“Amino acid difference” or “residue difference” refers to a change inthe amino acid residue at a position of a polypeptide sequence relativeto the amino acid residue at a corresponding position in a referencesequence. In some instances, residue differences are also referred to as“features” of the polypeptide sequence. The positions of amino aciddifferences generally are referred to herein as “Xn,” where n refers tothe corresponding position in the reference sequence upon which theresidue difference is based. For example, a “residue difference atposition X103 as compared to SEQ ID NO: 2” refers to a change of theamino acid residue at the polypeptide position corresponding to position103 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO: 2has a isoleucine at position 103, then a “residue difference at positionX103 as compared to SEQ ID NO:2” refers to an amino acid substitution ofany residue other than isoleucine at the position of the polypeptidecorresponding to position 103 of SEQ ID NO: 2. In most instances herein,the specific amino acid residue difference at a position is indicated as“XnY” where “Xn” specifies the corresponding position as describedabove, and “Y” is the single letter identifier of the amino acid foundin the engineered polypeptide (i.e., the different residue than in thereference polypeptide). In some embodiments, where more than one aminoacid can appear in a specified residue position, the alternative aminoacids can be listed in the form XnY/Z, where Y and Z represent alternateamino acid residues, or be presented as “Xn” followed by a list of thespecified residues. In some instances (e.g., in Table 2A, 2B, 2C, 2D and2E), the present disclosure also provides specific amino aciddifferences denoted by the conventional notation “AnB”, where A is thesingle letter identifier of the residue in the reference sequence, “n”is the number of the residue position in the reference sequence, and Bis the single letter identifier of the residue substitution in thesequence of the engineered polypeptide. The present disclosure includesengineered polypeptide sequences comprising one or more amino aciddifferences that include either/or both conservative andnon-conservative amino acid substitutions.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid, e.g., alanine, valine, leucine, and isoleucine; an amino acid withhydroxyl side chain is substituted with another amino acid with ahydroxyl side chain, e.g., serine and threonine; an amino acid havingaromatic side chains is substituted with another amino acid having anaromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, andhistidine; an amino acid with a basic side chain is substituted withanother amino acid with a basic side chain, e.g., lysine and arginine;an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain, e.g., aspartic acid or glutamicacid; and a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively. Exemplaryconservative substitutions are provided in Table 1 below.

TABLE 1 Residue Possible Conservative Substitutions A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) N, Q, S, T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C,P None

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine), (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered proline hydroxylase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered proline hydroxylase enzymes comprise insertionsof one or more amino acids to the naturally occurring prolinehydroxylase polypeptide as well as insertions of one or more amino acidsto other improved proline hydroxylase polypeptides. Insertions can be inthe internal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the reference sequence.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length proline hydroxylasepolypeptide, for example the polypeptide of SEQ ID NO:2 or engineeredpolypeptide of SEQ ID NO:34.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved proline hydroxylase enzymes may be presentwithin a cell, present in the cellular medium, or prepared in variousforms, such as lysates or isolated preparations. As such, in someembodiments, the improved proline hydroxylase enzyme can be an isolatedpolypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure proline hydroxylasecomposition will comprise about 60% or more, about 70% or more, about80% or more, about 90% or more, about 95% or more, and about 98% or moreof all macromolecular species by mole or % weight present in thecomposition. In some embodiments, the object species is purified toessential homogeneity (i.e., contaminant species cannot be detected inthe composition by conventional detection methods) wherein thecomposition consists essentially of a single macromolecular species.Solvent species, small molecules (<500 Daltons), and elemental ionspecies are not considered macromolecular species. In some embodiments,the isolated improved proline hydroxylases polypeptide is asubstantially pure polypeptide composition.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a chemical or enzymatic reaction thatis capable of converting a substrate, e.g., compound (2), to itscorresponding hydroxylated product, e.g., compound (1), with at leastabout 85% stereomeric excess.

“Regioselectivity” or “regioselective reaction” refers to a reaction inwhich one direction of bond making or breaking occurs preferentiallyover all other possible directions. Reactions can completely (100%)regioselective if the discrimination is complete, substantiallyregioselective (at least 75%), or partially regioselective (x %), if theproduct of reaction at one site predominates over the product ofreaction at other sites, for example, preferential formation of productcompound (1) over product compound (1a)).

“Improved enzyme property” refers to a proline hydroxylase polypeptidethat exhibits an improvement in any enzyme property as compared to areference proline hydroxylase. For the engineered proline hydroxylasepolypeptides described herein, the comparison is generally made to thewild-type proline hydroxylase enzyme, although in some embodiments, thereference proline hydroxylase can be another engineered prolinehydroxylase. Enzyme properties for which improvement is desirableinclude, but are not limited to, enzymatic activity (which can beexpressed in terms of percent conversion of the substrate), thermostability, solvent stability, pH activity profile, cofactorrequirements, refractoriness to inhibitors (e.g., substrate or productinhibition), and stereoselectivity.

“Increased enzymatic activity” refers to an improved property of theengineered proline hydroxylase polypeptides, which can be represented byan increase in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount of prolinehydroxylase) as compared to the reference proline hydroxylase enzyme.Exemplary methods to determine enzyme activity are provided in theExamples. Any property relating to enzyme activity may be affected,including the classical enzyme properties of K_(m), V_(max), or k_(cat),changes of which can lead to increased enzymatic activity. Improvementsin enzyme activity can be from about 1.2 times the enzymatic activity ofthe corresponding wild-type proline hydroxylase enzyme, to as much as 2times, 5 times, 10 times, 20 times, 25 times, 50 times or more enzymaticactivity than the naturally occurring proline hydroxylase or anotherengineered proline hydroxylase from which the proline hydroxylasepolypeptides were derived. Proline hydroxylase activity can be measuredby any one of standard assays, such as by monitoring changes inspectrophotometric properties of reactants or products. In someembodiments, the amount of products produced can be measured byHigh-Performance Liquid Chromatography (HPLC) separation combined withUV absorbance or fluorescent detection following derivatization, such aswith o-phthalaldehyde (OPA) or dansyl chloride. Comparisons of enzymeactivities are made using a defined preparation of enzyme, a definedassay under a set condition, and one or more defined substrates, asfurther described in detail herein. Generally, when lysates arecompared, the numbers of cells and the amount of protein assayed aredetermined as well as use of identical expression systems and identicalhost cells to minimize variations in amount of enzyme produced by thehost cells and present in the lysates.

“Conversion” refers to the enzymatic conversion of the substrate(s) tothe corresponding product(s). “Percent conversion” refers to the percentof the substrate that is converted to the product within a period oftime under specified conditions. Thus, the “enzymatic activity” or“activity” of a proline hydroxylase polypeptide can be expressed as“percent conversion” of the substrate to the product.

“Thermostable” refers to a proline hydroxylase polypeptide thatmaintains similar activity (more than 60% to 80% for example) afterexposure to elevated temperatures (e.g., 40-80° C.) for a period of time(e.g., 0.5-24 h) compared to the wild-type enzyme.

“Solvent stable” refers to a proline hydroxylase polypeptide thatmaintains similar activity (more than e.g., 60% to 80%) after exposureto varying concentrations (e.g., 5-99%) of solvent (ethanol, isopropylalcohol, dimethylsulfoxide (DMSO), tetrahydrofuran,2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyltert-butyl ether, etc.) for a period of time (e.g., 0.5-24 h) comparedto the wild-type enzyme.

“Thermo- and solvent stable” refers to a proline hydroxylase polypeptidethat is both thermostable and solvent stable.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m), values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad.Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychliket al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, NucleicAcids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered proline hydroxylase enzyme of the present disclosure.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m), as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theproline hydroxylase enzymes may be codon optimized for optimalproduction in the host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for many different organisms (see, e.g., Wada et al., 1992,Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res.28:292; Duret, et al., supra; Henaut and Danchin, “Escherichia coli andSalmonella,” 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C.,p. 2047-2066). The data source for obtaining codon usage may rely on anyavailable nucleotide sequence capable of coding for a protein. Thesedata sets include nucleic acid sequences actually known to encodeexpressed proteins (e.g., complete protein coding sequences-CDS),expressed sequence tags (ESTS), or predicted coding regions of genomicsequences (see for example, Mount, D., Bioinformatics: Sequence andGenome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol.266:259-281; Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the nucleic acid sequence encoding thepolypeptide. Such control sequences include, but are not limited to,promoter, transcription terminator a leader (i.e., translationinitiation) sequence, polyadenylation sequence, propeptide sequence, anda signal peptide sequence. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Suitable reaction conditions” refer to those conditions in thebiocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, co-substrate loading, cofactor loading, temperature,pH, buffers, co-solvents, etc.) under which a proline hydroxylasepolypeptide of the present disclosure is capable of converting asubstrate compound to a product compound (e.g., conversion of compound(2) to compound (1)). Exemplary “suitable reaction conditions” areprovided in the present disclosure and illustrated by the Examples.

“Loading”, such as in “compound loading” or “enzyme loading” or“cofactor loading” refers to the concentration or amount of a componentin a reaction mixture at the start of the reaction.

“Substrate” in the context of a biocatalyst mediated process refers tothe compound or molecule acted on by the biocatalyst. For example, anexemplary substrate for the proline hydroxylase biocatalyst in theprocess disclosed herein is compound (2).

“Product” in the context of a biocatalyst mediated process refers to thecompound or molecule resulting from the action of the biocatalyst. Forexample, an exemplary product for the proline hydroxylase biocatalyst inthe process disclosed herein is compound (1).

“Reductant” refers to a compound or agent capable of converting Fe⁺³ toFe⁺². An exemplary reductant is ascorbic acid, which is generally in theform of L-ascorbic acid.

“Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbonatoms inclusively, either straight chained or branched, more preferablyfrom 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbonatoms inclusively. An alkyl with a specified number of carbon atoms isdenoted in parenthesis, e.g., (C₁-C₆)alkyl refers to an alkyl of 1 to 6carbon atoms.

“Alkenyl” refers to hydrocarbon groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one doublebond but optionally containing more than one double bond.

“Alkynyl” refers to hydrocarbon groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one triplebond but optionally containing more than one triple bond, andadditionally optionally containing one or more double bonded moieties.

“Alkylene” refers to a straight or branched chain divalent hydrocarbonradical having from 1 to 18 carbon atoms inclusively, more preferablyfrom 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbonatoms inclusively, optionally substituted with one or more suitablesubstituents. Exemplary “alkylenes” include, but are not limited to,methylene, ethylene, propylene, butylene, and the like.

“Alkenylene” refers to a straight or branched chain divalent hydrocarbonradical having 2 to 12 carbon atoms inclusively and one or morecarbon-carbon double bonds, more preferably from 2 to 8 carbon atomsinclusively, and most preferably 2 to 6 carbon atoms inclusively,optionally substituted with one or more suitable substituents.

“Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer to alkyl,alkenyl and alkynyl as defined herein in which one or more of the carbonatoms are each independently replaced with the same or differentheteroatoms or heteroatomic groups. Heteroatoms and/or heteroatomicgroups which can replace the carbon atoms include, but are not limitedto, —O—, —S—, —S—O—, —NR^(γ)—, —PH—, —S(O)—, —S(O)₂—, —S(O)NR^(γ)—,—S(O)₂NR^(γ)—, and the like, including combinations thereof, where eachR¹ is independently selected from hydrogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

“Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to12 carbon atoms inclusively having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl). Exemplary arylsinclude phenyl, pyridyl, naphthyl and the like.

“Arylalkyl” refers to an alkyl substituted with an aryl, i.e.,aryl-alkyl-groups, preferably having from 1 to 6 carbon atomsinclusively in the alkyl moiety and from 6 to 12 carbon atomsinclusively in the aryl moiety. Such arylalkyl groups are exemplified bybenzyl, phenethyl and the like.

“Aryloxy” refers to —OR^(λ) groups, where R^(λ) is an aryl group, whichcan be optionally substituted.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 12 carbon atomsinclusively having a single cyclic ring or multiple condensed ringswhich can be optionally substituted with from 1 to 3 alkyl groups.Exemplary cycloalkyl groups include, but are not limited to, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl,1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and thelike, or multiple ring structures, including bridged ring systems, suchas adamantyl, and the like.

“Cycloalkylalkyl” refers to an alkyl substituted with a cycloalkyl,i.e., cycloalkyl-alkyl-groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 3 to 12 carbon atomsinclusively in the cycloalkyl moiety. Such cycloalkylalkyl groups areexemplified by cyclopropylmethyl, cyclohexylethyl and the like.

“Amino” refers to the group —NH₂. Substituted amino refers to the group—NHR^(η), NR^(η)R^(η), and NR^(η)R^(η)R^(η), where each R^(η) isindependently selected from substituted or unsubstituted alkyl,cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl,acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like.Typical amino groups include, but are limited to, dimethylamino,diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino,furanyl-oxy-sulfamino, and the like.

“Aminoalkyl” refers to an alkyl group in which one or more of thehydrogen atoms are replaced with one or more amino groups, includingsubstituted amino groups.

“Aminocarbonyl” refers to —C(O)NH₂. Substituted aminocarbonyl refers to—C(O)NR^(η)R^(η), where the amino group NR^(η)R^(η) is as definedherein.

“Oxy” refers to a divalent group —O—, which may have varioussubstituents to form different oxy groups, including ethers and esters.

“Alkoxy” or “alkyloxy” are used interchangeably herein to refer to thegroup —OR^(ζ), wherein R^(ζ) is an alkyl group, including optionallysubstituted alkyl groups.

“Carboxy” refers to —COOH.

“Carbonyl” refers to —C(O)—, which may have a variety of substituents toform different carbonyl groups including acids, acid halides, aldehydes,amides, esters, and ketones.

“Carboxyalkyl” refers to an alkyl in which one or more of the hydrogenatoms are replaced with one or more carboxy groups.

“Aminocarbonylalkyl” refers to an alkyl substituted with anaminocarbonyl group, as defined herein.

“Halogen” or “halo” refers to fluoro, chloro, bromo and iodo.

“Haloalkyl” refers to an alkyl group in which one or more of thehydrogen atoms are replaced with a halogen. Thus, the term “haloalkyl”is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. upto perhaloalkyls. For example, the expression “(C₁C₂) haloalkyl”includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl,1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl,perfluoroethyl, etc.

“Hydroxy” refers to —OH.

“Hydroxyalkyl” refers to an alkyl group in which in which one or more ofthe hydrogen atoms are replaced with one or more hydroxy groups.

“Thiol” or “sulfanyl” refers to —SH. Substituted thiol or sulfanylrefers to —S—R^(η), where R^(η) is an alkyl, aryl or other suitablesubstituent.

“Alkylthio” refers to —SR, where R is an alkyl, which can be optionallysubstituted. Exemplary alkylthio group include, but are not limited to,methylthio, ethylthio, n-propylthio, and the like.

“Arylthio” refers to —SR^(λ), where R^(λ) is an aryl, which can beoptionally substituted. Exemplary arylthio groups include, but are notlimited to, phenylthio, (4-methylphenyl)thio, pyridinylthio, and thelike.

“Alkylthioalkyl” refers to an alkyl substituted with an alkylthio group,—SR^(ζ), where R^(ζ) is an alkyl, which can be optionally substituted.

“Thiolalkyl” refers to an alkyl group in which one or more of thehydrogen atoms are replaced with one or more —SH groups.

“Sulfonyl” refers to —SO₂—. Substituted sulfonyl refers to —SO₂—R^(η),where R^(η) is an alkyl, aryl or other suitable substituent.

“Alkylsulfonyl” refers to —SO₂—R^(ζ), where R^(ζ) is an alkyl, which canbe optionally substituted. Typical alkylsulfonyl groups include, but arenot limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and thelike.

“Alkylsulfonylalkyl” refers to an alkyl substituted with analkylsulfonyl group, —SO₂—R^(ζ), where R is an alkyl, which can beoptionally substituted.

“Heteroaryl” refers to an aromatic heterocyclic group of from 1 to 10carbon atoms inclusively and 1 to 4 heteroatoms inclusively selectedfrom oxygen, nitrogen and sulfur within the ring. Such heteroaryl groupscan have a single ring (e.g., pyridyl or furyl) or multiple condensedrings (e.g., indolizinyl or benzothienyl).

“Heteroarylalkyl” refers to an alkyl substituted with a heteroaryl,i.e., heteroaryl-alkyl-groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 5 to 12 ring atomsinclusively in the heteroaryl moiety. Such heteroarylalkyl groups areexemplified by pyridylmethyl and the like.

“Heterocycle”, “heterocyclic” and interchangeably “heterocycloalkyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, from 2 to 10 carbon ring atoms inclusively andfrom 1 to 4 hetero ring atoms inclusively selected from nitrogen, sulfuror oxygen within the ring. Such heterocyclic groups can have a singlering (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings(e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples ofheterocycles include, but are not limited to, furan, thiophene,thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, pyrrolidine, indoline and the like.

“Heterocycloalkylalkyl” refers to an alkyl substituted with aheterocycloalkyl, i.e., heterocycloalkyl-alkyl-groups, preferably havingfrom 1 to 6 carbon atoms inclusively in the alkyl moiety and from 3 to12 ring atoms inclusively in the heterocycloalkyl moiety.

“Membered ring” is meant to embrace any cyclic structure. The numberpreceding the term “membered” denotes the number of skeletal atoms thatconstitute the ring. Thus, for example, cyclohexyl, pyridine, pyran andthiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, andthiophene are 5-membered rings.

“Fused bicyclic ring” as used herein refers to both unsubstituted andsubstituted carbocyclic and/or heterocyclic ring moieties having 5 to 8atoms in each ring, the rings having 2 common atoms.

Unless otherwise specified, positions occupied by hydrogen in theforegoing groups can be further substituted with substituentsexemplified by, but not limited to, hydroxy, oxo, nitro, methoxy,ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy,fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl,alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl,hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy,alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl,alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido,cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl,acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substitutedaryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl,heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl,substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl,morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; andpreferred heteroatoms are oxygen, nitrogen, and sulfur. It is understoodthat where open valences exist on these substituents they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further understood that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present disclosure, and is otherwise chemically reasonable.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. One of ordinary skill in the art would understand that withrespect to any molecule described as containing one or more optionalsubstituents, only sterically practical and/or synthetically feasiblecompounds are meant to be included. “Optionally substituted” refers toall subsequent modifiers in a term or series of chemical groups. Forexample, in the term “optionally substituted arylalkyl, the “alkyl”portion and the “aryl” portion of the molecule may or may not besubstituted, and for the series “optionally substituted alkyl,cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, andheteroaryl groups, independently of the others, may or may not besubstituted.

5.3 Engineered Proline Hydroxylase Polypeptides

The present disclosure provides polypeptides having proline hydroxylaseactivity, polynucleotides encoding the polypeptides, methods ofpreparing the polypeptides, and methods for using the polypeptides.Where the description relates to polypeptides, it is to be understoodthat it also describes the polynucleotides encoding the polypeptides.

Proline hydroxylases belong to a class of diooxygenase enzymes thatcatalyze hydroxylation of proline in presence of α-ketoglutarate andoxygen (O₂). The α-ketoglutarate is stoichiometrically decarboxylatedduring hydroxylation, with one atom of the O₂ molecule beingincorporated into the succinate and the other into the hydroxyl groupformed on the proline residue. As noted above, proline hydroxylases aredistinguished from prolyl hydroxylase by their ability to hydroxylatefree proline.

Several types of proline hydroxylases have been identified based on themajor diastereomeric products formed in the enzymatic reaction:cis-3-proline hydroxylase (cis-P3H), cis-4-proline hydroxylase(cis-P4H), trans-3-proline hydroxylase (trans-P3H), and trans-4-prolinehydroxylase (trans-P4H). cis-P3H enzymes have been identified inStreptomyces sp. TH1, Streptomyces canus and Bacillus sp. TH2 and TH3(Mori H. et al., 1996, Appl. Environ. Microbiol. 62 (6):1903-1907).trans-P3H has been identified in Glarea lozoyensis (Petersen, L. et al.,2003, Appl Microbiol Biotechnol. 62(2-3):263-7). cis-P4H have beenidentified in Lotus corniculatus rhizobia, Mesorhibozium loti,Sinorhizobium meliloti, and Medicago sativa rhizobia, (Hara and Kino,2009, Biochem Biophys Res Commun. 379(4):882-6; US Patent publicationno. 20110091942). trans-P4H have been identified in Dactylosporangiumsp., Amycolatopsis sp., Streptomyces griseoviridus, Streptomyces sp. andGlarea lozoyensis (Shibasaki T. et al., 1999, Appl. Environ. Microbiol65(9):4028-31; 2003, Petersen, L. et al., 2003, Appl MicrobiolBiotechnol. 62(2-3):263-7; Mori, H. et al., 1996, Appl. Environ.Microbiol. 62:1903-1907; Lawrence, C. C., et al., 1996, Biochem. J.313:185-191; and EP0641862).

The cis-4-proline hydroxylase from Sinorhizobium meliloti converts freeproline to the primary product cis-4-hydroxyproline. According to Kleinet al., supra, the enzyme also recognizes L-pipecolic acid, convertingit to a mixture of cis-5- and cis-3-hydroxypipecolic acid. However, theactivity on pipecolic acid is lower than on proline, and the enzyme isreported to have low specific activity and denature under reactionconditions (Klein et al., supra). Consequently, in vitro conversionreactions for preparing hydroxyproline and hydroxypipecolic acid with arecombinant wild-type enzyme expressed in E. coli was unsuitable as asynthetic strategy for commercial scale preparations. Whole cellsexpressing the enzyme was found to be more effective, but necessitatedthe use of defined growth medium lacking proline to minimize competitionby free proline and also simplify purification of the hydroxypipecolicacid product (Klein et al., supra).

In the present disclosure, engineered proline hydroxylases that overcomethe deficiencies of the wild-type cis-4-proline hydroxylase ofSinorhizobium meliloti are described. The engineered proline hydroxylasepolypeptides derived from the wild-type enzyme of Sinorhizobium melilotiare capable of efficiently converting in vitro free proline tocis-4-hydroxyproline, but also capable of efficiently converting a rangeof substrates, including the conversion of L-pipecolic acid (i.e.,(2S)-piperidine-2-carboxylic acid) to cis-5-hydroxypipecolic acid (i.e.,2S,5S)-5-hydroxypiperidine-2-carboxylic acid). Significantly, thepresent disclosure identifies amino acid residue positions andcorresponding mutations in the proline hydroxylase polypeptide sequencethat improve enzyme properties as compared to the naturally occurringenzyme, including among others, activity, stability, expression,regioselectivity, stereoselectivity, substrate tolerance, and substratespecificity. In particular, the present disclosure provides engineeredpolypeptides capable of efficiently converting substrate compound (2),(2S)-piperidine-2-carboxylic acid, to product compound (1),(2S,5S)-5-hydroxypiperidine-2-carboxylic acid (as illustrated in Scheme1 above) in presence of a co-substrate under suitable reactionconditions.

In some embodiments, the engineered proline hydroxylase polypeptidesshow increased activity in the conversion proline and(2S)-piperidine-2-carboxylic acid to product cis-4-hydroxy proline and(2S,5S)-5-hydroxypiperidine-2-carboxylic acid, respectively, in adefined time with the same amount of enzyme as compared to the wild-typeenzyme. In some embodiments, the engineered proline hydroxylasepolypeptide has at least about 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4fold, 5 fold, or 10 fold or more activity under suitable reactionconditions as compared to the polypeptide represented by SEQ ID NO:2.

In some embodiments, the engineered proline hydroxylase polypeptideshave increased regioselectivity as compared to the wild-type prolinehydroxylase. Specifically, the naturally occurring enzyme convertsproline to, primarily if not exclusively, cis-4-hydroxyproline, andconverts (2S)-piperidine-2-carboxylic acid to mixture of diastereomericproducts comprising compound (1),(2S,5S)-5-hydroxypiperidine-2-carboxylic acid and compound (1a),(2S,3R)-3-hydroxypiperidine-2-carboxylic acid. In some embodiments, theengineered proline hydroxylase polypeptides herein are capable ofselectively forming compound (1) in excess of product compound (1a). Insome embodiments, the engineered polypeptides are capable of selectivelyforming compound (1) in excess of product compound (1a), where the ratioof compound (1) formed over compound (1a) under suitable reactionconditions is at least 1.5, 2, 3, 4, 5, or 6 or more.

In some embodiments, the engineered proline hydroxylase polypeptides arecapable of converting the substrate compound (2) to product compound (1)without forming significant amounts of trans-5-hydroxypipecolic acid(i.e., (2S,5R)-5-hydroxypiperidine-2-carboxylic acid). In someembodiments, the engineered proline hydroxylase polypeptides are capableof converting the substrate compound (2) to product compound (1) undersuitable reaction conditions in greater than 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5 or greater diastereomeric excess of(2S,5R)-5-hydroxypiperidine-2-carboxylic acid.

In some embodiments, the engineered proline hydroxylase polypeptides arecapable of converting substrate compound (2) to product compound (1)under suitable reaction conditions with increased tolerance for thepresence of substrate relative to the reference polypeptide of SEQ IDNO: 2. Thus, in some embodiments the engineered proline hydroxylasepolypeptides are capable of converting substrate compound (2) to productcompound (1) at a substrate loading concentration of at least about 10g/L, about 20 g/L, about 30 g/L, about 40 g/L, about 50 g/L, about 70g/L, about 100 g/L, about 125 g/L, about 150 g/L. about 175 g/L or about200 g/L or more with a percent conversion of at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%, in a reaction time of about 120 h or less, 72 h orless, about 48 h or less, about 36 h or less, or about 24 h less, undersuitable reaction conditions.

The suitable reaction conditions under which the above-describedimproved properties of the engineered polypeptides carry out thehydroxylation reaction can be determined with respect to concentrationsor amounts of polypeptide, substrate, co-substrate, transition metalcofactor, reductant, buffer, co-solvent, pH, conditions includingtemperature and reaction time, and/or conditions with the polypeptideimmobilized on a solid support, as further described below and in theExamples.

The exemplary engineered polypeptides having proline hydroxylaseactivity with improved properties, particularly in the conversion ofcompound (2) to compound (1), comprises an amino acid sequence that hasone or more residue differences as compared to SEQ ID NO:2 at thefollowing residue positions: X2; X3; X4; X5; X9; X13; X17; X24; X25;X26; X29; X30; X36; X42; X52; X57; X58; X59; X62; X66; X86; X88; X92;X95; X98; X103; X112; X113; X114; X115; X116; X121; X131; X140; X150;X151; X166; X186; X188; X205; X225; X230; X270; and X271. The specificamino acid differences at each of these positions that are associatedwith the improved properties of the exemplary polypeptides of Tables 2A,2B, 2C, 2D, 2E, 2F, 2G, and 2H include: X2K; X2T; X3S; X4Q; X4L; X4E;X4S; X5I; X5L; X5M; X9I; X13T; X17V, X24R; X24S; X25R; X26R; X26T; X26W;X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G; X62Q; X66Q;X86S; X88R; X92V; X95M; X98F; X98T; X103L; X103Q; X112T; X112V; X113E;X114N; X115E; X115H; X115D; X115G; X115S; X115A; X116L; X121F; X131Y;X131F; X140L; X1505; X151A; X151H; X1515; X166T; X166L; X166Q; X186G;X188G; X205V; X225L; X225Y; X225W; X230V; X270E; X271K; and X271R.

The structure and function information for exemplary non-naturallyoccurring (or engineered) proline hydroxylase polypeptides of thepresent disclosure are based on the conversion of compound (2) tocompound (1), the results of which are shown below in Tables 2A, 2B, 2C,2D, 2E, 2F, 2G, and 2H. The odd numbered sequence identifiers (i.e., SEQID NOs) refer to the nucleotide sequence encoding the amino acidsequence provided by the even numbered SEQ ID NOs. The exemplarysequences are provided in the electronic sequence listing fileaccompanying this disclosure, which is hereby incorporated by referenceherein. The amino acid residue differences are based on comparison tothe reference sequence of SEQ ID NO: 2 (or SEQ ID NO: 4, or 6), whichrepresent the naturally occurring amino acid sequence of thecis-4-proline hydroxylase of Sinorhizobium meliloti. The activity ofeach engineered polypeptide relative to the reference polypeptide of SEQID NO: 2 was determined as conversion of the substrate,(2S)-piperidine-2-carboxylic acid, to product,(2S,5S)-5-hydroxypiperidine-2-carboxylic acid over a set time period andtemperature in a high-throughput (HTP) assay, which was used as theprimary screen. The HTP assay values in Tables 2A, 2B, and 2F weredetermined using E. coli. clear cell lysates in 96 well-plate format of˜200 μL volume per well following assay reaction conditions as noted inthe table and the Examples. In some instances, a shake flask powder(SFP) or downstream processed (DSP) powder assay was used as a secondaryscreen to assess the properties of the engineered proline hydroxylases,the results of which are provided in Tables 2C, 2D, 2E, 2G, and 2H. TheSFP forms provide a more purified powder preparation of the engineeredpolypeptides and can contain the engineered polypeptides that are up toabout 30% of total protein. The DSP preparations can provide an evenfurther purified form of the engineered polypeptide since thepreparations can contain the engineered proline hydroxylases that are upto about 80% of total protein. The engineered proline hydroxylases werealso examined for their regioselectivities by measuring the ratio(expressed as the selectivity ratio) of product compound (1),(2S,5S)-5-hydroxypiperidine-2-carboxylic acid, to product compound (1a),(2S,3R)-3-hydroxypiperidine-2-carboxylic acid, formed in the reactions.

TABLE 2A Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ ID NO: Amino Acid Differences Activity^(1,2)(Condition A) (nt/aa) (relative to SEQ ID NO: 2) (Relative to SEQ ID NO:2) 1/2 N/A 1   3/4 N/A 1³  5/6 N/A 1³  7/8 A42E 1.53  9/10 I103L 2.3711/12 I103Q 2.26 13/14 F116L 1.15 15/16 N131Y 1.7  17/18 A150S 2.0719/20 S29A; H166T 1.47 21/22 H166T 1.90 23/24 H166Q 4.96 25/26 S30V 1.3327/28 S30P 2.75 29/30 A36T 1.8  31/32 S59G 1.54 33/34 V57T 1.54 35/36Q52P; S225Y 1.3  37/38 V57A 1.43 39/40 T115H 1.68 41/42 L112T 1.87 43/44A66Q 1.21 45/46 H271K 1.24 47/48 T115D 1.25 49/50 T115G; L121F 1.4 51/52 H271R 1.31 53/54 L112V 1.32 55/56 T115S 1.31 57/58 V25R; V58A 1.4959/60 S2K 1.55 61/62 S2T 1.52 63/64 E13T 1.6  65/66 H4Q 6.27 67/68 H4L2.44 69/70 F5I 2.32 71/72 F5L 3.9  73/74 F5M 1.94 75/76 V9I 2.81 77/78H4E 1.28 79/80 H4L; T115A 1.22 81/82 H4S 1.75 83/84 S225W 1.47 85/86S225L 1.6  87/88 L230V 1.28 89/90 N131F 1.26 91/92 T92V 1.37 93/94 V95M1.73 95/96 T115G 1.37 97/98 T3S 6.43 ¹HTP Assay Condition A (see alsoExample 4): Cells grown in 96 well plates were lysed with 100 μL LysisBuffer (1 mg/mL lysozyme, 0.5 mg/mL polymyxin B sulfate (PMBS), and 50mM phosphate buffer, pH 6.3). The reaction conditions for a 200 μLreaction comprised: 10 g/L substrate compound (2); 19 g/L α-ketoglutaricacid; 21 g/L L-ascorbic acid; 1.5 mM Mohr's salt; 50 mM potassiumphosphate buffer, pH 6.3 (pH adjusted with KOH); 100 μL crude lysate;and reaction temperature at about 25° C. (room temperature) for about 24h. Plates were covered with an O₂ permeable seal and shaken on atitre-plate shaker at speed #2.5. ²Activity relative to SEQ ID NO: 2 iscalculated as the % conversion of the product formed per % conversion ofthe corresponding SEQ ID NO: 2 under the reaction conditions specified.% Conversion was quantified by firstly dividing the areas of the productpeak by the sum of the areas of the substrate, product andimpurities/side product peak as determined by HPLC analysis and thensubtracting it with the % conversion of the negative control(overlapping peak). ³The amino acid sequences of polypeptidesrepresented by SEQ ID NO: 4 and 6 are identical to the naturallyoccurring proline hydroxylase amino acid sequence of SEQ ID NO: 2, butwhich were prepared in E. coli using the differently codon-optimizedgenes of SEQ ID NO: 3 and 5, respectively. The codon optimized genes ofSEQ ID NO: 3 and 5 exhibited at 1.2-fold increased expression of theproline hydroxylase polypeptide in E. coli as compared to the expressionof SEQ ID NO: 1.

TABLE 2B Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ ID Activity (Condition B)¹ NO: Amino AcidDifferences (relative to (nt/aa) (relative to SEQ ID NO: 2) SEQ ID NO:112)² 113/114 C86S; T92V; I103L; M151S; H166Q; 2.3 G270E 115/116 I103Q;F116L; H166L; S225L 1.53 117/118 A66Q; I103L; H166Q 1.48 119/120 A66Q;I103L; H166Q 1.81 121/122 A66Q; T92V; I103L; H166Q 1.67 123/124 T92V;I103L; H166Q 1.51 125/126 A66Q; T92V; I103L; D113E; T115S; 1.86 H166Q127/128 V25R; A66Q; T92V; I103L; T115E; 1.54 H166Q ¹HTP Assay ConditionB (see also Example 4): Cells grown in 96 well plates were lysed with100 μL Lysis Buffer (1 mg/mL lysozyme, 0.5 mg/mL polymyxin B sulfate(PMBS), and 50 mM phosphate buffer, pH 6.3). The reaction conditions fora 200 μL reaction comprised: 10 g/L substrate compound (2); 19 g/Lα-ketoglutaric acid; 21 g/L ascorbic acid; 1.5 mM Mohr's salt; 50 mMphosphate buffer, pH 6.3 (pH adjusted with KOH); 100 μL crude lysate;and a reaction temperature of c.a. 25° C. (room temperature) for aboutc.a. 24 h. The plates were covered by an O₂ permeable seal, and theplates shaken on a titre-plate shaker at speed #2.5. ²Activity relativeto SEQ ID NO: 112 is calculated as the % conversion of the productformed as compared to the % conversion of the corresponding SEQ ID NO:112 under the reaction conditions specified. % Conversion was quantifiedby firstly dividing the areas of the product peak by the sum of theareas of the substrate, product and impurities/side product peak asdetermined by HPLC analysis and then subtracting it with the %conversion of the negative control (overlapping peak).

TABLE 2C Engineered Polypeptides and Relative Enzyme Improvements Using“Mini-DSP” Enzyme Preparations SEQ ID Selectivity NO: Amino AcidDifferences Ratio: (nt/aa) (relative to SEQ ID NO: 2) % Conv¹ (1)/(1a)²1/2 N/A 16.6³ 1.6 3/4 N/A 49.6³ 2.1  9/10 I103L 21.2³ 5 23/24 H166Q53.7³ 2.5  99/100 H166Q 51.8³ 2.4 101/102 I103L 24.5³ 3.1 103/104 I103L;H166Q 67.3³ 5.5 105/106 I103L; N131Y; H166Q 40³   5.7 107/108 T3S;I103L; H166Q 66.9⁴ 5.2 129/130 T3S; A66Q; T92V; I103L; H166Q 67.8⁴ 5.1131/132 T3S; V25R; A66Q; T92V; I103L; 79.7⁴ 5.6 T115E; H166Q 133/134T3S; V25R; A66Q; T92V; I103L; 69.9⁴ 5.1 H166Q ¹% Conversion wasquantified by dividing the area of the product peak by the sum of theareas of the substrate compound (2), product compound (1) and productcompound (1a) peak as determined by HPLC analysis. ²Selectivity ratiorefers to the ratio of HPLC areas for product compound (1) over productcompound (1a). ³Reaction Condition D was used: Reaction was carried outin a 2 mL reaction vessel. Reaction mixture comprised: 10 g/L compound(2); 19 g/L α-ketoglutaric acid; 21 g/L L-ascorbic acid; 1.5 mM Mohr'ssalt; 50 mM potassium phosphate buffer, pH 6.3; 20 g/L protein ofMini-DSP powder preparation; and a reaction temperature of about c.a.25° C. (room temperature) for about c.a. 24 h. Reactions were stirred at1200 rpm and reaction vial kept open to the atmosphere. ⁴ReactionCondition E was used: Reaction was carried out in a 250 mL two neckround bottom flask. Initial mixture of 60 mL of 50 mM potassiumphosphate buffer was charged with 0.146 g (1.5 g/L) of Mohr's salt(ammonium iron[II] sulfate hexahydrate), 3.46 g (35 g/L) ofα-ketoglutaric acid and 1.36 g (14 g/L) of ascorbic acid with pHadjusted to 6.3 with 50% KOH. Enzyme was added to mixture as 0.2 g (2g/L) mini DSP preparation pre-dissolved in 16 mL 50 mM potassiumphosphate buffer, pH 6.3, with 1 mL antifoam, with stirring for 15minutes. Substrate was added to the mixture as 2 g (20 g/L) of compound(2) (pre-dissolved in 24 mL 50 mM potassium phosphate buffer, pH 6.3)with stirring at 25° C. under oxygen sparging (flow rate = 2 L/h).

TABLE 2D Engineered Polypeptides and Relative Enzyme Improvements UsingFull DSP Preparations Amino Acid Condition D¹ Condition F² Condition E⁴SEQ ID Differences Selectivity Selectivity Selectivity NO: (relative toRatio Ratio Ratio (nt/aa) SEQ ID NO: 2) % Conv⁵ (1)/(1a)⁶ % Conv⁵(1)/(1a)⁶ % Conv⁵ (1)/(1a)⁶ 1/2 N/A 83.5 5.7 46.4 1.9 — — 103/104 I103L;H166Q 86.7 8.1 71.3 4.7 — — 107/108 T3S; I103L; — — 74.7 6 — — H166Q109/110 A26T; I103L; — — 73.5 4.6 — — H166Q 131/132 T3S; V25R; — — — —66.9 6.5:1 A66Q; T92V; I103L; T115E; H166Q ¹Reaction Condition D wasused: Reaction was carried out in a 2 mL reaction vessel. Reactionmixture comprised: 10 g/L substrate compound (2); 19 g/L α-ketoglutaricacid; 21 g/L L-ascorbic acid; 1.5 mM Mohr's salt; 50 mM potassiumphosphate buffer, pH 6.3; 20 g/L Full DSP enzyme powder; and a reactiontemperature of about 25° C. for about 24 h. Reactions were stirred at1200 rpm, and the vial kept open to the atmosphere. ²Reaction ConditionF was used: Reaction was carried out in a 1 mL reaction vessel. Reactionmixture comprised: 20 g/L substrate compound (2); 38 g/L α-ketoglutaricacid; 21 g/L L-ascorbic acid; 1.5 mM Mohr's salt; 50 mM potassiumphosphate buffer, pH 6.3; 10 g/L Full DSP enzyme powder; and a reactiontemperature of about 25° C. for c.a. 24 h. Reactions were stirred at1200 rpm, and the vial kept open to the atmosphere. ⁴DSP ReactionCondition E was used: Reaction was carried out in a 250 mL two neckround bottom flask. Initial mixture of 60 mL of 50 mM potassiumphosphate buffer was charged with 0.146 g (1.5 g/L) of Mohr's salt(ammonium iron[II] sulfate hexahydrate), 3.46 g (35 g/L) ofα-ketoglutaric acid and 1.36 g (14 g/L) of L-ascorbic acid with pHadjusted to 6.3 with 50% KOH. Enzyme was added to the mixture as 0.2 g(2 g/L) Full DSP powder pre-dissolved in 16 mL 50 mM potassium phosphatebuffer, pH 6.3, with 1 mL antifoam, with stirring for 15 minutes.Substrate was added to the mixture as 2 g (20 g/L) of compound (2)(pre-dissolved in 24 mL 50 mM phosphate buffer, pH 6.3) with stirring at25° C. under oxygen sparging (flow rate = 2 L/h). ⁵% Conversion wasquantified by dividing the area of the product peak by the sum of theareas of the substrate, product compound (1) and product compound (1a)peak as determined by HPLC analysis. ⁶Selectivity ratio refers to theratio of HPLC areas for product compound (1) over the product compound(1a).

TABLE 2E Engineered Polypeptides and Relative Enzyme Improvements UsingSFP Preparations SEQ ID Selectivity NO: Amino Acid Differences Activ-Ratio (nt/aa) (relative to SEQ ID NO: 2) ity¹ % Conv⁴ (1)/(1a)⁴ 111/112I103L; H166Q 0.19² 3.9 3.1 125/126 A66Q; T92V; I103L; D113E; 1.8³ 11.13.1 T115S; H166Q 127/128 V25R; A66Q; T92V; I103L; 1.8³ 5.9 3.5 T115E;H166Q ¹SFP Reaction Condition C was used: Reaction was carried out in a200 μL reaction vessel. Reaction mixture comprised: 30 g/L substratecompound (2); 52.5 g/L α-ketoglutaric acid; 21 g/L L-ascorbic acid; 2.25mM Mohr's salt; 50 mM potassium phosphate buffer, pH 6.3 (pH adjustedwith KOH); 5 g/L protein of SFP enzyme powder preparation; and areaction temperature of c.a. 25° C. for 24 h. Plates were covered withan O₂ permeable seal and shaken on a litre-plate shaker at speed #2.5.²Activity relative to SEQ ID NO: 108 is calculated as the % conversionof the product formed as compared to the % conversion of thecorresponding SEQ ID NO: 108 under the reaction conditions specified. %Conversion was quantified by firstly dividing the areas of the productpeak by the sum of the areas of the substrate, product andimpurities/side product peak as determined by HPLC analysis and thensubtracting it with the % conversion of the negative control(overlapping peak). ³Activity relative to SEQ ID NO: 124 is calculatedas the % conversion of the product formed per % conversion of thecorresponding SEQ ID NO: 124 under the reaction conditions specified. %Conversion was quantified by firstly dividing the areas of the productpeak by the sum of the areas of the substrate, product andimpurities/side product peak as determined by HPLC ⁴Reaction conditionsused for % Conv and Selectivity Ratio differed from SFP ReactionConditions above as follows. Reaction in a 100 mL vessel. Reactionmixture comprised: 20 g/L of substrate compound (2); 2 g/L of SFP enzymepowder preparation; 14 g/L of ascorbic acid; 35 g/L ketoglutaric acid;1.5 g/L Mohr's salt; 1% (v/v) antifoam; and oxygen sparging at 2 L/h.

TABLE 2F Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations Fold- improved SEQ ID Fold- activity² Fold- NO: AminoAcid Differences improved (+ascorbic improved (nt/aa) (relative to SEQID NO: 2) activity¹ acid preinc.)² selectivity³ 137/138 V25R; A66Q;T92V; I103L; E114N; T115E; 1.5 n.d. 1 H166Q; 139/140 V25R; A66Q; T92V;I103L; T115E; M140L; 1.7 n.d. 1.1 H166Q; 141/142 V25R; A66Q; T92V; S98T;I103L; T115E; 4.1 n.d. 1.1 H166Q; 143/144 V25R; A42L; A66Q; T92V; I103L;T115E; 0.8 n.d. 1.3 H166Q; 145/146 A17V; V25R; A26W; A66Q; T92V; I103L;3.8 n.d. 0.8 T115E; H166Q; 147/148 V25R; A62Q; A66Q; T92V; I103L; T115E;1.5 n.d. 0.9 H166Q; 149/150 V25R; A26R; A66Q; T92V; I103L; T115E; 1.7n.d. 0.9 H166Q; 151/152 V25R; A66Q; T92V; I103L; T115E; M151A; 1.4 n.d.1 H166Q; 153/154 V25R; A66Q; A88R; T92V; I103L; T115E; 1.4 n.d. 0.9H166Q; 155/156 V25R; A66Q; T92V; I103L; T115E; H166Q; 4.4 n.d. 0.9V188G; 157/158 E24R; V25R; A66Q; T92V; I103L; T115E; 1.5 n.d. 0.9 H166Q;159/160 E24S; V25R; A66Q; T92V; I103L; T115E; 1.6 n.d. 0.9 H166Q;161/162 V25R; A66Q; T92V; S98F; I103L; T115E; 0.9 n.d. 1.3 H166Q;163/164 T3S; V25R; A42L; A66Q; T92V; I103L; T115E; + n.d. + H166Q;165/166 T3S; V25R; A42L; A66Q; T92V; S98T; I103L; + n.d. + T115E; H166Q;167/168 T3S; V25R; A42L; A66Q; T92V; S98T; I103L; + n.d. + T115E; H166Q;V188G; 169/170 E24S; V25R; A42L; A66Q; T92V; I103L; T115E; 1.43 1.664.46 H166Q; V188G; 171/172 E24S; V25R; A42L; A62Q; A66Q; T92V; S98T;1.60 1.54 4.30 I103L; E114N; T115E; M140L; H166Q; 173/174 E24S; V25R;A42L; A66Q; T92V; S98T; I103L; 1.77 2.41 5.92 E114N; T115E; H166Q;175/176 E24S; V25R; A26R; A42L; A66Q; T92V; I103L; 1.73 1.70 4.50 T115E;H166Q; 177/178 V25R; A42L; A62Q; A66Q; T92V; I103L; T115E; 1.41 1.624.98 M140L; H166Q; V188G; 179/180 E24S; V25R; A26R; A42L; A66Q; A88R;T92V; 2.59 1.81 3.94 I103L; T115E; M140L; H166Q; 181/182 E24S; V25R;A26R; A42L; A66Q; A88R; T92V; 1.68 1.41 4.31 S98T; I103L; E114N; T115E;M140L; H166Q; V188G; 183/184 E24S; V25R; A42L; A62Q; A66Q; T92V; I103L;1.46 3.13 4.84 E114N; T115E; M140L; H166Q; 185/186 E24S; V25R; A26R;A42L; A66Q; A88R; T92V; 1.71 2.08 4.17 I103L; E114N; T115E; H166Q;187/188 E24S; V25R; A42L; A66Q; T92V; I103L; T115E; 1.68 2.11 4.47M140L; H166Q; 189/190 E24S; V25R; A42L; A66Q; T92V; S98T; I103L; + 1.584.21 E114N; T115E; H166Q; 191/192 V25R; A26R; A42L; A66Q; T92V; I103L;1.87 1.53 3.77 E114N; T115E; M151H; H166Q; 193/194 E24S; V25R; A42L;A62Q; A66Q; T92V; S98T; 2.19 1.53 4.08 I103L; T115E; M140L; M151H;H166Q; 195/196 V25R; A42L; A66Q; T92V; I103L; E114N; 1.62 1.87 5.45T115E; H166Q; 197/198 V25R; A42L; A62Q; A66Q; T92V; I103L; T115E; + 1.733.74 M140L; M151H; H166Q; I205V; 199/200 E24S; V25R; A42L; A66Q; A88R;T92V; I103L; 1.51 2.29 4.32 E114N; T115E; H166Q; 201/202 E24S; V25R;A26R; A42L; A62Q; A66Q; T92V; 1.16 1.85 5.34 I103L; T115E; M151H; H166Q;203/204 E24S; V25R; A26R; A42L; A66Q; A88R; T92V; 2.02 2.01 4.11 I103L;E114N; T115E; H166Q; 205/206 E24S; V25R; A26R; A42L; A62Q; A66Q; T92V; +1.58 3.76 I103L; T115E; M140L; M151H; H166Q; 207/208 E24S; V25R; A26R;A42L; A66Q; A88R; T92V; + 1.50 3.59 I103L; E114N; T115E; H166Q; Q186G;209/210 E24S; V25R; A26W; A42L; A66Q; A88R; T92V; 1.23 1.50 3.64 I103L;T115E; M140L; H166Q; 211/212 E24S; V25R; A26R; A42L; A62Q; A66Q; A88R; +2.28 3.95 T92V; I103L; E114N; T115E; M140L; M151H; H166Q; Q186G; 213/214E24S; V25R; A42L; A66Q; T92V; S98F; I103L; + 1.51 6.2 T115E; M140L;H166Q; V188G; 215/216 E24S; V25R; A26R; A42L; A62Q; A66Q; A88R; + 1.326.3 T92V; S98F; I103L; E114S; T115E; M140L; M151H; H166Q; 217/218 E24S;V25R; A26R; A42L; A66Q; T92V; S98F; + 1.33 6.5 I103L; T115E; M140L;M151H; H166Q; 219/220 E24S; V25R; A42L; A66Q; T92V; S98F; I103L; + 1.538.8 T115E; H166Q; 221/222 T3S; E24S; V25R; A42L; A66Q; T92V; S98T; +n.d. + I103L; E114N; T115E; H166Q; 223/224 T3S; E24S; V25R; A26R; A42L;A66Q; A88R; + n.d. + T92V; I103L; T115E; M140L; H166Q; 225/226 T3S;E24S; V25R; A42L; A62Q; A66Q; T92V; + n.d. + I103L; E114N; T115E; M140L;H166Q; 227/228 T3S; E24S; V25R; A26R; A42L; A66Q; A88R; + n.d. + T92V;I103L; T115E; M140L; H166Q; ¹HTP Assay - Conditions F For polypeptidesof SEQ ID NO: 138-168: Cells grown in 96 well plates were lysed with 100μL Lysis Buffer (1 mg/mL lysozyme, 0.5 mg/mL polymyxin B sulfate (PMBS),and 50 mM phosphate buffer, pH 6.3). The reaction conditions for a 200μL reaction comprised: 30 g/L substrate compound (2); 52.5 g/Lα-ketoglutaric acid; 21 g/L L-ascorbic acid; 2.25 g/L Mohr's salt; 50 mMpotassium phosphate buffer, pH 6.3 (pH adjusted with KOH); 100 μL crudelysate; and reaction temperature at about 25° C. (room temperature) forabout 24 h. Plates were covered with an O₂ permeable seal and shaken ona titre-plate shaker at speed #2.5. Activity was calculated as the %conversion of the product formed as compared to the % conversion of thecorresponding reference polypeptide of SEQ ID NO: 128 under the reactionconditions specified. % Conversion was quantified by firstly dividingthe areas of the product peak by the sum of the areas of the substrate,product and impurities/side product peak as determined by HPLC analysisand then subtracting it with the % conversion of the negative control(overlapping peak). For polypeptides of SEQ ID NO: 170-228: Same as forSEQ ID NO: 138-168 except reaction temperature at 30° C. Activity wascalculated as the % conversion of the product formed as compared to the% conversion of the corresponding reference polypeptide of SEQ ID NO:144 under the reaction conditions specified. % Conversion was quantifiedby firstly dividing the areas of the product peak by the sum of theareas of the substrate, product and impurities/side product peak asdetermined by HPLC analysis and then subtracting it with the %conversion of the negative control (overlapping peak). ²HTP AssayConditions G - Ascorbic Acid Preincubation HTP assays of thepolypeptides of SEQ ID NO: 170-228 also were carried out following 2 hhour preincubation of the lysate in ascorbic acid. Preincubationprocedure: 100 μL of lysate was transferred into a new deep-well platefollowed by the addition of 20 μL/well of ascorbic acid stock solution.The plate was shaken at room temperature, speed 2.5 for 2 h. HTPactivity assay was carried out using the HTP Assay Conditions at 30° C.for SEQ ID NO: 170-228 as described above. ³Improved Selectivity:Improved selectivity for the 2,5-regioisomer product of compound (1)versus the 2,3-regioisomer product of compound (1a) produced in the HTPAssays carried out under Condition F was determined using LC-MS. LC-MSSample Preparation: 10 μL of HTP Assay reaction mixture was pipetted outand diluted twenty thousand fold (in three stages) with 1:1 mixture ofMeCN:water. The sample was vortexed and spun down at 12,000 r.p.m. for 5minutes. The supernatant was transferred into a 2 mL vial for LC-MSanalysis using analysis parameters described in Example 7. “+” indicatesactivity or selectivity improved from 1-fold to 5-fold relative to SEQID NO: 2. “n.d.” indicates “not determined”

TABLE 2G Engineered Polypeptides and Relative Enzyme Improvements UsingSFP Preparations SEQ ID Fold- Selectivity NO: Amino Acid Differencesimproved % Ratio (nt/aa) (relative to SEQ ID NO: 2) activity¹conversion² (1)/(1a)³ 127/128 V25R; A66Q; T92V; I103L; T115E; H166Q 18.9 3.9:1 137/138 V25R; A66Q; T92V; I103L; E114N; T115E; 0.9 8.2 3.2:1H166Q; 139/140 V25R; A66Q; T92V; I103L; T115E; M140L; 1.2 10.4 3.4:1H166Q; 141/142 V25R; A66Q; T92V; S98T; I103L; T115E; 1.9 16.7 3.5:1H166Q; 143/144 V25R; A42L; A66Q; T92V; I103L; T115E; 1.3 11.8 4.5:1H166Q; 173/174 E24S; V25R; A42L; A66Q; T92V; S98T; I103L; 1.8 8.9 3.9:1E114N; T115E; H166Q; 179/180 E24S; V25R; A26R; A42L; A66Q; A88R; T92V;2.9 8.2 3.2:1 I103L; T115E; M140L; H166Q; 183/184 E24S; V25R; A42L;A62Q; A66Q; T92V; I103L; 1.8 10.4 3.4:1 E114N; T115E; M140L; H166Q;185/186 E24S; V25R; A26R; A42L; A66Q; A88R; T92V; 1.9 16.7 3.5 1 I103L;E114N; T115E; H166Q; 187/188 E24S; V25R; A42L; A66Q; T92V; I103L; T115E;1.2 11.8 4.5:1 M140L; H166Q; 199/200 E24S; V25R; A42L; A66Q; A88R; T92V;I103L; 1.7 8.9 3.9:1 E114N; T115E; H166Q; 203/204 E24S; V25R; A26R;A42L; A66Q; A88R; T92V; 1.8 8.2 3.2:1 I103L; E114N; T115E; H166Q; ¹SFPActivity Assay - Conditions G This assay was carried out on a 5 mL scalewith final reaction mixture conditions: 10 g/L SFP of polypeptide, 30g/L substrate of compound (2), 52.5 g/L α-ketoglutaric acid, 21 g/Lascorbic acid, 2.25 g/L Mohr's salt in 50 mM phosphate buffer pH 6.3. Apremix stock solution consisting of the following was prepared: 37.5 g/L(187.5 mg) substrate, 65.63 g/L (328 mg) α-ketoglutaric acid, and 26.25g/L (131.3 mg) ascorbic acid. After pH was adjusted 2.81 g/L (14 mg) ofMohr's salt at were added and dissolved in 5 mL of 50 mM phosphatebuffer, pH 6.3 (for each vial reaction) and pH was adjusted to pH 6.3with 50% KOH. A 50 g/L SFP enzyme stock solution was prepared bydissolving 100 mg SFP preparation of the desired polypeptide variant in2 mL of 50 mM phosphate buffer pH 6.3. The assay was initiated by adding4 mL of the premix stock solution into a glass vial followed by 1 mL ofenzyme stock solution. The vial was stirred at 250 rpm, room temperatureusing a magnetic hot plate stirrer for 24 h. 10 mL of 75% acetonitrile(MeCN) in water was added to quench the reaction with stirring. 1 mL ofthe quenched reaction was then taken to a Costar 96 deep well 2 mL assayblock and spun down for 10 min at 4000 r.p.m. Dansylation and HPLCanalysis was carried out as described for SFP assays in Example 3.Activity was calculated as the % conversion of the product formed ascompared to the % conversion of the corresponding reference polypeptideof SEQ ID NO: 128 under the reaction conditions specified. % Conversionwas quantified by firstly dividing the areas of the product peak by thesum of the areas of the substrate, product and impurities/side productpeak as determined by HPLC analysis and then subtracting it with the %conversion of the negative control (overlapping peak). ²% Conversion wasquantified by dividing the area of the product peak by the sum of theareas of the substrate compound (2), product compound (1) and productcompound (1a) peak as determined by HPLC analysis. ³Selectivity ratiorefers to the ratio of HPLC areas for product compound (1) over productcompound (1a).

TABLE 2H Engineered Polypeptides and Relative Enzyme Improvements UsingDSP Preparations SEQ ID Selectivity NO: Amino Acid Differences Ratio(nt/aa) (relative to SEQ ID NO: 2) % conversion³ (1)/(1a)⁴ Condition H¹131/132 T3S; V25R; A66Q; T92V; I103L; T115E; H166Q 30.0 5.1:1 163/164T3S; V25R; A42L; A66Q; T92V; I103L; T115E; H166Q; 21.9 8.6:1 165/166T3S; V25R; A42L; A66Q; T92V; S98T; I103L; T115E; 21.0 9.4:1 H166Q;167/168 T3S; V25R; A42L; A66Q; T92V; S98T; I103L; T115E; 18.3 9.7:1H166Q; V188G; 221/222 T3S; E24S; V25R; A42L; A66Q; T92V; S98T; I103L;21.0  9:1 E114N; T115E; H166Q; 223/224 T3S; E24S; V25R; A26R; A42L;A66Q; A88R; T92V; 33.0 6.7:1 I103L; T115E; M140L; H166Q; 225/226 T3S;E24S; V25R; A42L; A62Q; A66Q; T92V; I103L; 24.9 6.5:1 E114N; T115E;M140L; H166Q; Condition I² 223/224 T3S; E24S; V25R; A26R; A42L; A66Q;A88R; T92V; 51.7 6.7:1 I103L; T115E; M140L; H166Q; 227/228 T3S; E24S;V25R; A26R; A42L; A66Q; A88R; T92V; 77.0² 6.7:1 I103L; T115E; M140L;H166Q; ¹DSP Assay - Condition H: This DSP assay was carried out on a 10mL scale. A solution of substrate of compound (2) (0.30 g) in 2 ml of 50mM potassium phosphate buffer (pH 6.3) was prepared and added to apre-stirred mixture of DSP polypeptide enzyme (0.05 g), antifoam Y-30emulsion (100 to 300 μL), Mohr's salt (0.021 g), α-ketoglutaric acid(0.51 g) and ascorbic acid (0.204 g) in 7 mL of 50 mM potassiumphosphate buffer (pH 6.3). The resulting mixture was continuouslysparged with oxygen (2 L/h) at stirred 25° C. for 24 h. Reactionprogress was monitored by HPLC as described in Example 6. ²DSP Assay -Condition I: This DSP assay was carried out on a 100 mL scale. Asolution of substrate of compound (2) (3.0 g) in 20 ml of 50 mMpotassium phosphate buffer (pH 6.3) was prepared and added to apre-stirred mixture of DSP polypeptide enzyme (0.5 g), antifoam Y-30emulsion (1 mL), Mohr's salt (0.21 g), α-ketoglutaric acid (5.1 g) andascorbic acid (2.04 g) in 70 mL of 50 mM potassium phosphate buffer (pH6.3). The resulting mixture was continuously sparged with oxygen (2 L/h)and stirred at 25° C. for 24 h. Reaction progress was monitored by HPLCas described in Example 6. ³% Conversion was quantified by dividing thearea of the product peak by the sum of the areas of the substratecompound (2), product compound (1) and product compound (1a) peak asdetermined by HPLC analysis. ⁴Selectivity ratio refers to the ratio ofHPLC areas for product compound (1) over product compound (1a).

From an analysis of the exemplary polypeptides, improvements in enzymeproperties are associated with residue differences as compared to SEQ IDNO:2 at residue positions X2; X3; X4; X5; X9; X13; X17; X24; X25; X26;X29; X30; X36; X42; X52; X57; X58; X59; X62; X66; X86; X88; X92; X95;X98; X103; X112; X113; X114; X115; X116; X121; X131; X140; X150; X151;X166; X186; X188; X205; X225; X230; X270; and X271. The specific residuedifferences at each of these positions that are associated with theimproved properties include: X2K; X2T; X3S; X4Q; X4L; X4E; X4S; X5I;X5L; X5M; X9I; X13T; X17V, X24R; X24S; X25R; X26R; X26T; X26W; X29A;X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G; X62Q; X66Q; X86S;X88R; X92V; X95M; X98F; X98T; X103L; X103Q; X112T; X112V; X113E; X114N;X115E; X115H; X115D; X115G; X115S; X115A; X116L; X121F; X131Y; X131F;X140L; X150S; X151A; X151H; X151S; X166T; X166L; X166Q; X186G; X188G;X205V; X225L; X225Y; X225W; X230V; X270E; X271K; and X271R.

The specific enzyme properties associated with the residues differencesas compared to SEQ ID NO:2 at the residue positions above include, amongothers, enzyme activity, regioselectivity, polypeptide expression, andsubstrate tolerance. Improvements in enzyme activity and substratetolerance are associated with residue differences at residue positionsX3; X17; X24; X25; X26; X29; X30; X36; X42; X52; X57; X58; X59; X62;X66; X86; X88; X92; X95; X103; X112; X113; X114; X115; X116; X121; X131;X140; X150; X151; X166; X188; X225; X230; X270; and X271. Improvementsin regioselectivity are associated with residue differences at residuepositions: X3; X25; X42; X66; X92; X98; X103; X115; X131; and X166.Improvements in polypeptide expression are associated with residuedifferences at residue positions: X2; X4; X5; X9; and X13. Accordingly,the residue differences at the foregoing residue positions can be usedindividually or in various combinations to produce engineered prolinehydroxylase polypeptides having the desired improved properties,including, among others, enzyme activity, regioselectivity,stereoselectivity, and substrate tolerance. Other residue differencesaffecting polypeptide expression can be used to increase expression ofthe engineered proline hydroxylase.

In light of the guidance provided herein, it is further contemplatedthat any of the exemplary engineered polypeptides of SEQ ID NO: 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and 228can be used as the starting amino acid sequence for synthesizing otherengineered proline hydroxylase polypeptides, for example by subsequentrounds of evolution that incorporate new combinations of various aminoacid differences from other polypeptides in Tables 2A, 2B, 2C, 2D, 2E,2F, 2G, and 2H, and other residue positions described herein. Furtherimprovements may be generated by including amino acid differences atresidue positions that had been maintained as unchanged throughoutearlier rounds of evolution.

Accordingly, in some embodiments, the engineered polypeptide havingproline hydroxylase activity comprises an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to reference sequence SEQ IDNO:2 and one or more residue differences as compared to SEQ ID NO:2 atresidue positions selected from: X2; X3; X4; X5; X9; X13; X17; X24; X25;X26; X29; X30; X36; X42; X52; X57; X58; X59; X62; X66; X86; X88; X92;X95; X98; X103; X112; X113; X114; X115; X116; X121; X131; X140; X150;X151; X166; X186; X188; X205; X225; X230; X270; and X271.

In some embodiments, the engineered polypeptide having prolinehydroxylase activity with improved properties as compared to SEQ IDNO:2, comprises an amino acid sequence having at least 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentity to a reference sequence selected from SEQ ID NO: 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and 228, andone or more residue differences as compared to SEQ ID NO:2 at residuepositions selected from: X2; X3; X4; X5; X9; X13; X17; X24; X25; X26;X29; X30; X36; X42; X52; X57; X58; X59; X62; X66; X86; X88; X92; X95;X98; X103; X112; X113; X114; X115; X116; X121; X131; X140; X150; X151;X166; X186; X188; X205; X225; X230; X270; and X271. In some embodiments,the reference amino acid sequence is selected from SEQ ID NO: 10, 24,104, 106, 108, 110, 132, 164, 222, 224, 226, and 228. In someembodiments, the reference amino acid sequence is SEQ ID NO:10. In someembodiments, the reference amino acid sequence is SEQ ID NO:24. In someembodiments, the reference amino acid sequence is SEQ ID NO:104. In someembodiments, the reference amino acid sequence is SEQ ID NO:108. In someembodiments, the reference amino acid sequence is SEQ ID NO:110. In someembodiments, the reference amino acid sequence is SEQ ID NO:132. In someembodiments, the reference amino acid sequence is SEQ ID NO:164. In someembodiments, the reference amino acid sequence is SEQ ID NO:222. In someembodiments, the reference amino acid sequence is SEQ ID NO:224. In someembodiments, the reference amino acid sequence is SEQ ID NO:226. In someembodiments, the reference amino acid sequence is SEQ ID NO:228.

In some embodiments, the engineered polypeptide having prolinehydroxylase activity with improved properties as compared to SEQ IDNO:2, comprises an amino acid sequence selected from SEQ ID NO: 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and228, and having one or more residue differences as compared to SEQ IDNO:2 at residue positions selected from: X2; X3; X4; X5; X9; X13; X17;X24; X25; X26; X29; X30; X36; X42; X52; X57; X58; X59; X62; X66; X86;X88; X92; X95; X98; X103; X112; X113; X114; X115; X116; X121; X131;X140; X150; X151; X166; X186; X188; X205; X225; X230; X270; and X271. Insome embodiments, the amino acid sequence is selected from SEQ ID NO:10, 24, 104, 106, 108, 110, 132, 164, 222, 224, 226, and 228. In someembodiments, the amino acid sequence is SEQ ID NO:10. In someembodiments, the amino acid sequence is SEQ ID NO:24. In someembodiments, the amino acid sequence is SEQ ID NO:104. In someembodiments, the amino acid sequence is SEQ ID NO:108. In someembodiments, the amino acid sequence is SEQ ID NO:110. In someembodiments, the amino acid sequence is SEQ ID NO:132. In someembodiments, the reference amino acid sequence is SEQ ID NO:164. In someembodiments, the reference amino acid sequence is SEQ ID NO:222. In someembodiments, the reference amino acid sequence is SEQ ID NO:224. In someembodiments, the reference amino acid sequence is SEQ ID NO:226. In someembodiments, the reference amino acid sequence is SEQ ID NO:228.

In some embodiments, the residue differences at residue positions X2;X3; X4; X5; X9; X13; X17; X24; X25; X26; X29; X30; X36; X42; X52; X57;X58; X59; X62; X66; X86; X88; X92; X95; X98; X103; X112; X113; X114;X115; X116; X121; X131; X140; X150; X151; X166; X186; X188; X205; X225;X230; X270; and X271 are selected from X2K; X2T; X3S; X4Q; X4L; X4E;X4S; X5I; X5L; X5M; X9I; X13T; X17V, X24R; X24S; X25R; X26R; X26T; X26W;X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G; X62Q; X66Q;X86S; X88R; X92V; X95M; X98F; X98T; X103L; X103Q; X112T; X112V; X113E;X114N; X115E; X115H; X115D; X115G; X1155; X115A; X116L; X121F; X131Y;X131F; X140L; X1505; X151A; X151H; X1515; X166T; X166L; X166Q; X186G;X188G; X205V; X225L; X225Y; X225W; X230V; X270E; X271K; and X271R.

Accordingly, in some embodiments, the engineered proline hydroxylasepolypeptides displaying one or more of the improved properties describedherein can comprise an amino acid sequence having the amino acidsequence identity to a reference sequence as described above, and one ormore residue differences as compared to SEQ ID NO:2 selected from: X2K;X2T; X3S; X4Q; X4L; X4E; X4S; X5I; X5L; X5M; X9I; X13T; X17V, X24R;X24S; X25R; X26R; X26T; X26W; X29A; X30V; X30P; X36T; X42E; X52P; X57T;X57A; X58A; X59G; X62Q; X66Q; X86S; X88R; X92V; X95M; X98F; X98T; X103L;X103Q; X112T; X112V; X113E; X114N; X115E; X115H; X115D; X115G; X1155;X115A; X116L; X121F; X131Y; X131F; X140L; X1505; X151A; X151H; X1515;X166T; X166L; X166Q; X186G; X188G; X205V; X225L; X225Y; X225W; X230V;X270E; X271K; and X271R.

In some embodiments, the engineered proline hydroxylase has an aminoacid sequence comprising at least one or more residue differences ascompared to SEQ ID NO:2 selected from: X25R; X26T; X103L; X115E;X131Y/F; and X166Q.

In some embodiments, the engineered proline hydroxylase polypeptidecomprises an amino acid sequence having at least a combination ofresidues differences as compared to SEQ ID NO:2 selected from: (a) X103Land X166Q; (b) X52P and X255Y; (c) X4E/L/S and X115A; (d) X25R and X58A;(e) X29A and X166T/Q/L; (f) X115H/D/G and X121F; (g) X3S, X103L, andX166Q; (h) X103L, X131Y/F, and X166T/Q/L; (i) X26T, X103L and X166T/Q/L;(j) X25R, X66Q, X92V and X115E; (k) X25R, X66Q, X92V, X103L, X115E, andX166Q; and (l) X3S, X25R, X66Q, X92V, X103L, X115E, and X166Q.

As will be appreciated by the skilled artisan, in some embodiments, oneor a combination of residue differences above that is selected can bekept constant (i.e., maintained) in the engineered proline hydroxylasesas a core feature, and additional residue differences at other residuepositions incorporated into the sequence to generate additionalengineered proline hydroxylase polypeptides with improved properties.Accordingly, it is to be understood for any engineered prolinehydroxylase containing one or a subset of the residue differences above,the present disclosure contemplates other engineered prolinehydroxylases that comprise the one or subset of the residue differences,and additionally one or more residue differences at the other residuepositions disclosed herein. By way of example and not limitation, anengineered proline hydroxylase comprising a residue difference atresidue position X103, can further incorporate one or more residuedifferences at the other residue positions, e.g., X2; X3; X4; X5; X9;X13; X17; X24; X25; X26; X29; X30; X36; X42; X52; X57; X58; X59; X62;X66; X86; X88; X92; X95; X98; X112; X113; X114; X115; X116; X121; X131;X140; X150; X151; X166; X186; X188; X205; X225; X230; X270; and X271.Another example is an engineered proline hydroxylase comprising aresidue difference at residue position X166, which can further compriseone or more residue differences at the other residue positions, e.g.,X2; X3; X4; X5; X9; X13; X17; X24; X25; X26; X29; X30; X36; X42; X52;X57; X58; X59; X62; X66; X86; X88; X92; X95; X98; X103; X112; X113;X114; X115; X116; X121; X131; X140; X150; X151; X186; X188; X205; X225;X230; X270; and X271.

As noted above, the engineered polypeptides having proline hydroxylaseactivity are also capable of converting substrate compound (2) toproduct compound (1). In some embodiments, the engineered prolinehydroxylase polypeptide is capable of converting the substrate compound(2) to the product compound (1) with at least 1.2 fold, 1.5 fold, 2fold, 3 fold, 4 fold, 5 fold, 10 fold or more activity relative to theactivity of the reference polypeptide of SEQ ID NO: 2. In someembodiments, the engineered proline hydroxylase polypeptide capable ofconverting the substrate compound (2) to the product compound (1) withat least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold ormore activity relative to the activity of the reference polypeptide ofSEQ ID NO:2 comprises an amino acid sequence having one or more featuresselected from: X2K; X2T; X3S; X4Q; X4L; X4E; X4S; X5I; X5L; X5M; X9I;X13T; X17V, X24R; X24S; X25R; X26R; X26T; X26W; X29A; X30V; X30P; X36T;X42E; X52P; X57T; X57A; X58A; X59G; X62Q; X66Q; X86S; X88R; X92V; X95M;X98F; X98T; X103L; X103Q; X112T; X112V; X113E; X114N; X115E; X115H;X115D; X115G; X1155; X115A; X116L; X121F; X131Y; X131F; X140L; X1505;X151A; X151H; X1515; X166T; X166L; X166Q; X186G; X188G; X205V; X225L;X225Y; X225W; X230V; X270E; X271K; and X271R.

In some embodiments, the engineered proline hydroxylase polypeptide iscapable of converting the substrate compound (2) to the product compound(1) with at least 1.2 fold the activity relative to SEQ ID NO:2 andcomprises an amino acid sequence selected from: SEQ ID NO: 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, and 228.

In some embodiments, the engineered proline hydroxylase polypeptide iscapable of converting the substrate compound (2) to the product compound(1) with at least 2 fold the activity relative to SEQ ID NO:2 andcomprises an amino acid sequence having one or more residue differencesselected from: X3S; X30P; X86S; X103L; X103Q; X113E; X115E; X1505;X166Q; X1515; X225L; and 270E.

In some embodiments, the engineered proline hydroxylase polypeptidecapable of converting the substrate compound (2) to the product compound(1) with at least 2 fold the activity relative to SEQ ID NO:2 comprisesan amino acid sequence selected from: SEQ ID NO: 10, 12, 18, 24, 28, 66,68, 70, 72, 76, 98, 100, 102, 104, 106, 108, 110; 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and 228.

In some embodiments, the engineered proline hydroxylase polypeptide iscapable of converting at least 50% or more, 60% or more, 70% or more,89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% ormore, or 95% or more of compound (2) to compound (1) in 120 h or less,72 h or less, 48 h or less, or 24 or less, at a substrate loading ofabout 100 g/L, about 50 g/L, or about 20 g/L under HTP Assay conditions,under SFP Assay conditions, or DSP Assay conditions. In someembodiments, the engineered proline hydroxylase polypeptide is capableof converting at least 50% or more of compound (2) to compound (1) in 24h or less at a substrate loading of about 20 g/L under DSP Assayconditions at about 25° C.

In some embodiments, the engineered proline hydroxylase polypeptide iscapable of converting substrate compound (2) to product compound (1) inexcess of compound (1a). The residue differences identified in theexemplary engineered proline hydroxylases of Tables 2A, 2B, 2C, 2D, 2E,2F, 2G, and 2H are shown to maintain or increase the regioselectivityfor compound (1) over compound (1a) in the conversion reaction. In someembodiments, the engineered proline hydroxylase polypeptides are capableof converting substrate compound (2) to product compound (1) in excessof compound (1a), where the ratio of compound (1) formed over compound(1a) is at least a ratio of 1.5, 2, 3, 4, 5, or 6 or more, particularlyunder HTP Assay, SFP Assay, or DSP Assay conditions.

In some embodiments, the engineered proline hydroxylase polypeptidecapable of converting substrate compound (2) to product compound (1) inexcess of compound (1a) in at least a ratio of 2 or greater comprises anamino acid sequence have at least one or more of the following features:X103L; X115E; X166Q and X131Y. In some embodiments, the engineeredproline hydroxylase capable of converting substrate compound (2) toproduct compound (1) in excess of compound (1a) in at least a ratio of 2or greater comprises an amino acid sequence selected from SEQ ID NO: 10,24, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, and 228.

In some embodiments, the engineered proline hydroxylase polypeptidecapable of converting substrate compound (2) to product compound (1) inexcess of compound (1a) in at least a ratio of 4 or greater comprises anamino acid sequence have at least the features X103L and X166Q. In someembodiments, the engineered proline hydroxylase capable of convertingsubstrate compound (2) to product compound (1) in excess of compound(1a) in at least a ratio of 4 or greater comprises an amino acidsequence selected from SEQ ID NO: 104, 106, 108, 110, 130, 132 134, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, and 228.

In some embodiments, the engineered proline hydroxylase is capable ofconverting substrate compound (2) to product compound (1) in stereomericexcess of compound (1R), (2S,5R)-5-hydroxypiperidine-2-carboxylic acid,

The wild-type enzyme is characterized by its ability to convert(2S)-piperidine-2-carboxylic acid to(2S,5S)-5-hydroxypiperidine-2-carboxylic acid, with little if any of thetrans hydroxy product (1R). As shown herein, the residue differences inthe exemplary engineered proline hydroxylase polypeptides of Tables 2A,2B, 2C, 2D, 2E, 2F, 2G, and 2H maintain the high diastereoselectivity,including those polypeptides with non-conservative changes to the aminoacid sequence. In some embodiments, the engineered proline hydroxylasecapable of converting substrate compound (2) to product compound (1) indiasstereomeric excess of compound (1R) comprises an amino acid sequencehaving one or more features selected from: X2K; X2T; X3S; X4Q; X4L; X4E;X4S; X5I; X5L; X5M; X9I; X13T; X17V, X24R; X24S; X25R; X26R; X26T; X26W;X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G; X62Q; X66Q;X86S; X88R; X92V; X95M; X98F; X98T; X103L; X103Q; X112T; X112V; X113E;X114N; X115E; X115H; X115D; X115G; X115S; X115A; X116L; X121F; X131Y;X131F; X140L; X150S; X151A; X151H; X151S; X166T; X166L; X166Q; X186G;X188G; X205V; X225L; X225Y; X225W; X230V; X270E; X271K; and X271R. Insome embodiments, the product compound (1) is formed in at least 90%,95%, 96%, 97%, 98%, 99%, or greater diastereomeric excess of thecompound (1R). In some embodiments, no detectable amount of transhydroxy product (1R) is formed by the engineered polypeptides undersuitable reaction conditions.

In some embodiments, the engineered proline hydroxylase has an aminoacid sequence comprising one or more residue differences as compared toSEQ ID NO:2 that increase expression of the engineered prolinehydroxylase activity in a bacterial host cell, particularly in E. coli.In some embodiments, the amino acid sequence that show increasedexpression in a bacterial host cell comprises one or more residuedifferences selected from: X2K; X2T; X4Q; X4L; X4E; X4S; X5I; X5L; X5M;X9I; and X13T.

In some embodiments, the engineered proline hydroxylase polypeptide withimproved properties in the conversion of compound (2) to compound (1)has an amino acid sequence comprising a sequence selected from SEQ IDNO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, and 228.

In some embodiments, the engineered polypeptide having prolinehydroxylase activity, comprises an amino acid sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity to one of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, and 228, and the amino acidresidue differences as compared to SEQ ID NO:2 present in any one of SEQID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, and 228, as provided in Tables 2A, 2B, 2C, 2D, 2E, 2F, 2G, and2H.

In addition to the residue positions specified above, any of theengineered proline hydroxylase polypeptides disclosed herein can furthercomprise other residue differences relative to SEQ ID NO:2 at otherresidue positions, i.e., residue positions other than X2; X3; X4; X5;X9; X13; X17; X24; X25; X26; X29; X30; X36; X42; X52; X57; X58; X59;X62; X66; X86; X88; X92; X95; X98; X103; X112; X113; X114; X115; X116;X121; X131; X140; X150; X151; X166; X186; X188; X205; X225; X230; X270;and X271. Residue differences at these other residue positions canprovide for additional variations in the amino acid sequence withoutadversely affecting the ability of the polypeptide to carry out theconversion of proline to cis-4-hydroxyproline as well as conversion ofcompound (2) to compound (1). Accordingly, in some embodiments, inaddition to the amino acid residue differences present in any one of theengineered proline hydroxylase polypeptides selected from SEQ ID NO: 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,and 228, the sequence can further comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24,1-26, 1-30, 1-35, 1-40, 1-45, or 1-50 residue differences at other aminoacid residue positions as compared to the SEQ ID NO:2. In someembodiments, the number of amino acid residue differences as compared tothe reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50residue positions. In some embodiments, the number of amino acid residuedifferences as compared to the reference sequence can be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25residue positions. The residue difference at these other positions canbe conservative changes or non-conservative changes. In someembodiments, the residue differences can comprise conservativesubstitutions and non-conservative substitutions as compared to thenaturally occurring proline hydroxylase polypeptide of SEQ ID NO: 2.

In some embodiments, the present disclosure also provides engineeredpolypeptides that comprise a fragment of any of the engineered prolinehydroxylase polypeptides described herein that retains the functionalactivity and/or improved property of that engineered prolinehydroxylase. Accordingly, in some embodiments, the present disclosureprovides a polypeptide fragment capable of converting compound (2) tocompound (1) under suitable reaction conditions, wherein the fragmentcomprises at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% of afull-length amino acid sequence of an engineered proline hydroxylasepolypeptide of the present disclosure, such as an exemplary engineeredproline hydroxylase polypeptide selected from SEQ ID NO: 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and 228.

In some embodiments, the engineered proline hydroxylase polypeptide canhave an amino acid sequence comprising a deletion of any one of theengineered proline hydroxylase polypeptides described herein, such asthe exemplary engineered polypeptides of SEQ ID NO: 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and 228. Thus,for each and every embodiment of the engineered proline hydroxylasepolypeptides of the disclosure, the amino acid sequence can comprisedeletions of one or more amino acids, 2 or more amino acids, 3 or moreamino acids, 4 or more amino acids, 5 or more amino acids, 6 or moreamino acids, 8 or more amino acids, 10 or more amino acids, 15 or moreamino acids, or 20 or more amino acids, up to 10% of the total number ofamino acids, up to 10% of the total number of amino acids, up to 20% ofthe total number of amino acids, or up to 30% of the total number ofamino acids of the proline hydroxylase polypeptides, where theassociated functional activity and/or improved properties of theengineered proline hydroxylase described herein are maintained. In someembodiments, the deletions can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35,1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the numberof deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 aminoacid residues. In some embodiments, the deletions can comprise deletionsof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21,22, 23, 24, or 25 amino acid residues.

In some embodiments, the engineered proline hydroxylase polypeptideherein can have an amino acid sequence comprising an insertion ascompared to any one of the engineered proline hydroxylase polypeptidesdescribed herein, such as the exemplary engineered polypeptides of SEQID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, and 228. Thus, for each and every embodiment of the prolinehydroxylase polypeptides of the disclosure, the insertions can compriseone or more amino acids, 2 or more amino acids, 3 or more amino acids, 4or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 ormore amino acids, 10 or more amino acids, 15 or more amino acids, 20 ormore amino acids, 30 or more amino acids, 40 or more amino acids, or 50or more amino acids, where the associated functional activity and/orimproved properties of the engineered proline hydroxylase describedherein is maintained. The insertions can be to amino or carboxyterminus, or internal portions of the proline hydroxylase polypeptide.

In some embodiments, the engineered proline hydroxylase polypeptideherein can have an amino acid sequence comprising a sequence selectedfrom SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,220, 222, 224, 226, and 228, and optionally one or several (e.g., up to3, 4, 5, or up to 10) amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the number of amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

In the above embodiments, the suitable reaction conditions for theengineered polypeptides can be those described in Tables 2A, 2B, 2C, 2D,2E, 2F, 2G, and 2H. Accordingly, in some embodiments, the suitablereaction conditions are HTP Assay conditions, which can comprise: 10 g/Lor 20 g/L substrate compound loading; 19 g/L α-ketoglutaric acid; 21 g/LL-ascorbic acid; 1.5 mM Mohr's salt; 50 mM potassium phosphate buffer,pH 6.3 (pH adjusted with KOH); 100 μL crude lysate; and a reactiontemperature at about 25° C. (room temperature) for a reaction time ofabout 24 h.

In some embodiments, the suitable reaction conditions are thosedescribed for shake flask powder (SFP) assays, which can comprise: 30g/L substrate compound loading; 52.5 g/L α-ketoglutaric acid; 21 g/LL-ascorbic acid; 2.25 mM Mohr's salt; 50 mM potassium phosphate buffer,pH 6.3 (pH adjusted with KOH); 5 g/L protein of SFP enzyme powderpreparation; and a reaction temperature of c.a. 25° C. (roomtemperature) for a reaction time of about 24 h.

In some embodiments, the suitable reaction conditions are thosedescribed for mini downstream process powder (DSP) assays, whichcomprise: 10 g/L or 20 g/L substrate loading; 19 g/L α-ketoglutaricacid; 21 g/L L-ascorbic acid; 1.5 mM Mohr's salt; 50 mM potassiumphosphate buffer, pH 6.3; 20 g/L protein of DSP powder preparation; anda reaction temperature of about 25° C. (room temperature) for aboutreaction time of about 24 h.

Guidance for use of these foregoing reaction conditions and the prolinehydroxylase polypeptides are given in, among others, Tables 2A, 2B, 2C,2D, 2E, 2F, 2G, and 2H, and the Examples.

In some embodiments, the polypeptides of the disclosure can be in theform of fusion polypeptides in which the engineered polypeptides arefused to other polypeptides, such as, by way of example and notlimitation, antibody tags (e.g., myc epitope), purification sequences(e.g., His tags for binding to metals), and cell localization signals(e.g., secretion signals). Thus, the engineered polypeptides describedherein can be used with or without fusions to other polypeptides.

It is to be understood that the polypeptides described herein are notrestricted to the genetically encoded amino acids. In addition to thegenetically encoded amino acids, the polypeptides described herein maybe comprised, either in whole or in part, of naturally occurring and/orsynthetic non-encoded amino acids. Certain commonly encounterednon-encoded amino acids of which the polypeptides described herein maybe comprised include, but are not limited to: the D-stereomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine(Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Nat); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Pat); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aGly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

In some embodiments, the engineered polypeptides can be in variousforms, for example, such as an isolated preparation, as a substantiallypurified enzyme, whole cells transformed with gene(s) encoding theenzyme, and/or as cell extracts and/or lysates of such cells. Theenzymes can be lyophilized, spray-dried, precipitated or be in the formof a crude paste, as further discussed below.

In some embodiments, the engineered polypeptides can be provided on asolid support, such as a membrane, resin, solid carrier, or other solidphase material. A solid support can be composed of organic polymers suchas polystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of a solid support can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation.

In some embodiments, the engineered polypeptides having prolinehydroxylase activity of the present disclosure can be immobilized on asolid support such that they retain their improved activity,stereoselectivity, and/or other improved properties relative to thereference polypeptide of SEQ ID NO: 2. In such embodiments, theimmobilized polypeptides can facilitate the biocatalytic conversion ofthe substrate compounds of formula (II), (VI) or other suitablesubstrates, to the product compound of formula (I), (V), orcorresponding products, respectively (e.g., as shown in Schemes 1, 2 and3), and after the reaction is complete are easily retained (e.g., byretaining beads on which polypeptide is immobilized) and then reused orrecycled in subsequent reactions. Such immobilized enzyme processesallow for further efficiency and cost reduction. Accordingly, it isfurther contemplated that any of the methods of using the prolinehydroxylase polypeptides of the present disclosure can be carried outusing the same proline hydroxylase polypeptides bound or immobilized ona solid support.

Methods of enzyme immobilization are well-known in the art. Theengineered polypeptides can be bound non-covalently or covalently.Various methods for conjugation and immobilization of enzymes to solidsupports (e.g., resins, membranes, beads, glass, etc.) are well known inthe art and described in e.g., Yi et al., “Covalent immobilization ofto-transaminase from Vibrio fluvialis JS17 on chitosan beads,” ProcessBiochemistry 42(5): 895-898 (May 2007); Martin et al., “Characterizationof free and immobilized (S)-aminotransferase for acetophenoneproduction,” Applied Microbiology and Biotechnology 76(4): 843-851(September 2007); Koszelewski et al., “Immobilization of ω-transaminasesby encapsulation in a sol-gel/celite matrix,” Journal of MolecularCatalysis B: Enzymatic, 63: 39-44 (April 2010); Truppo et al.,“Development of an Improved Immobilized CAL-B for the EnzymaticResolution of a Key Intermediate to Odanacatib,” Organic ProcessResearch & Development, published online: dx.doi.org/10.1021/op200157c;Hermanson, G. T., Bioconjugate Techniques, Second Edition, AcademicPress (2008); Mateo et al., “Epoxy sepabeads: a novel epoxy support forstabilization of industrial enzymes via very intense multipoint covalentattachment,” Biotechnology Progress 18(3):629-34 (2002); andBioconjugation Protocols: Strategies and Methods, In Methods inMolecular Biology, C. M. Niemeyer ed., Humana Press (2004); thedisclosures of each which are incorporated by reference herein. Solidsupports useful for immobilizing the engineered proline hydroxylases ofthe present disclosure include but are not limited to beads or resinscomprising polymethacrylate with epoxide functional groups,polymethacrylate with amino epoxide functional groups, styrene/DVBcopolymer or polymethacrylate with octadecyl functional groups.Exemplary solid supports useful for immobilizing the engineered prolinehydroxylase polypeptides of the present disclosure include, but are notlimited to, chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi),including the following different types of SEPABEAD: EC-EP, EC-HFA/S,EXA252, EXE119 and EXE120.

In some embodiments, the polypeptides described herein can be providedin the form of kits. The enzymes in the kits may be present individuallyor as a plurality of enzymes. The kits can further include reagents forcarrying out the enzymatic reactions, substrates for assessing theactivity of enzymes, as well as reagents for detecting the products. Thekits can also include reagent dispensers and instructions for use of thekits.

In some embodiments, the kits of the present disclosure include arrayscomprising a plurality of different proline hydroxylase polypeptides atdifferent addressable position, wherein the different polypeptides aredifferent variants of a reference sequence each having at least onedifferent improved enzyme property. In some embodiments, a plurality ofpolypeptides immobilized on solid supports can be configured on an arrayat various locations, addressable for robotic delivery of reagents, orby detection methods and/or instruments. The array can be used to test avariety of substrate compounds for conversion by the polypeptides. Sucharrays comprising a plurality of engineered polypeptides and methods oftheir use are described in, e.g., WO2009/008908A2.

5.4 Polynucleotides Encoding Engineered Proline Hydroxylases, ExpressionVectors and Host Cells

In another aspect, the present disclosure provides polynucleotidesencoding the engineered proline hydroxylase polypeptides describedherein. The polynucleotides may be operatively linked to one or moreheterologous regulatory sequences that control gene expression to createa recombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered proline hydroxylase can be introduced into appropriatehost cells to express the corresponding proline hydroxylase polypeptide.

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode the improved proline hydroxylase enzymes. Thus,having knowledge of a particular amino acid sequence, those skilled inthe art could make any number of different nucleic acids by simplymodifying the sequence of one or more codons in a way which does notchange the amino acid sequence of the protein. In this regard, thepresent disclosure specifically contemplates each and every possiblevariation of polynucleotides that could be made encoding thepolypeptides described herein by selecting combinations based on thepossible codon choices, and all such variations are to be consideredspecifically disclosed for any polypeptide described herein, includingthe amino acid sequences presented in Tables 2A, 2B, 2C, 2D, 2E, 2F, 2G,and 2H and disclosed in the sequence listing incorporated by referenceherein as SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, and 228.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. In some embodiments, all codons need not be replaced to optimizethe codon usage of the proline hydroxylases since the natural sequencewill comprise preferred codons and because use of preferred codons maynot be required for all amino acid residues. Consequently, codonoptimized polynucleotides encoding the proline hydroxylase enzymes maycontain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greaterthan 90% of codon positions of the full length coding region.

In some embodiments, the polynucleotide comprises a codon optimizednucleotide sequence encoding the naturally occurring proline hydroxylasepolypeptide amino acid sequence, as represented by SEQ ID NO:2. In someembodiments, the polynucleotide has a nucleic acid sequence comprisingat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identity to the codon optimized nucleic acid sequences of SEQ IDNO: 1, 3, or 5, each of which encodes the identical polypeptidesequences of SEQ ID NO:2, 4, or 6, respectively. The codon optimizedsequences of SEQ ID NO:1, 3, or 5 enhance expression of the encoded,wild-type proline hydroxylase, providing preparations of enzyme capableof converting in vitro over 80% of compound (2) to compound (1) undermini-DSP Assay conditions, and converting over 45% of compound (2) tocompound (1) under DSP Assay conditions. In some embodiments, the codonoptimized polynucleotide sequence can enhance expression of the prolinehydroxylase by at least 1.2 fold, 1.5 fold or 2 fold or greater ascompared to the naturally occurring polynucleotide sequence fromSinorhizobium meliloti, which is disclosed herein as SEQ ID NO: 135.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference sequence of SEQ ID NO:1, 3, or 5, or a complement thereof, and encodes a polypeptide havingproline hydroxylase activity.

In some embodiments, as described above, the polynucleotide encodes anengineered polypeptide having proline hydroxylase activity with improvedproperties as compared to SEQ ID NO: 2, where the polypeptide comprisesan amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to areference sequence selected from SEQ ID NO: 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, and 228, and one or moreresidue differences as compared to SEQ ID NO:2 selected from: X2K; X2T;X3S; X4Q; X4L; X4E; X4S; X5I; X5L; X5M; X9I; X13T; X17V, X24R; X24S;X25R; X26R; X26T; X26W; X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A;X58A; X59G; X62Q; X66Q; X86S; X88R; X92V; X95M; X98F; X98T; X103L;X103Q; X112T; X112V; X113E; X114N; X115E; X115H; X115D; X115G; X1155;X115A; X116L; X121F; X131Y; X131F; X140L; X1505; X151A; X151H; X1515;X166T; X166L; X166Q; X186G; X188G; X205V; X225L; X225Y; X225W; X230V;X270E; X271K; and X271R. In some embodiments, the reference amino acidsequence is selected from SEQ ID NO: 10, 24, 104, 106, 108, 110, 132,164, 222, 224, 226, and 228. In some embodiments, the reference aminoacid sequence is SEQ ID NO:10. In some embodiments, the reference aminoacid sequence is SEQ ID NO:24. In some embodiments, the reference aminoacid sequence is SEQ ID NO:104. In some embodiments, the reference aminoacid sequence is SEQ ID NO:108. In some embodiments, the reference aminoacid sequence is SEQ ID NO:110. In some embodiments, the reference aminoacid sequence is SEQ ID NO:132. In some embodiments, the reference aminoacid sequence is SEQ ID NO:164. In some embodiments, the reference aminoacid sequence is SEQ ID NO:222. In some embodiments, the reference aminoacid sequence is SEQ ID NO:224. In some embodiments, the reference aminoacid sequence is SEQ ID NO:226. In some embodiments, the reference aminoacid sequence is SEQ ID NO:228.

In some embodiments, the polynucleotide encodes a proline hydroxylasepolypeptide capable of converting substrate compound (2) to productcompound (1) with improved properties as compared to SEQ ID NO:2,wherein the polypeptide comprises an amino acid sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to reference sequence SEQ ID NO:2 andone or more residue differences as compared to SEQ ID NO: 2 at residuepositions selected from: X2; X3; X4; X5; X9; X13; X17; X24; X25; X26;X29; X30; X36; X42; X52; X57; X58; X59; X62; X66; X86; X88; X92; X95;X98; X103; X112; X113; X114; X115; X116; X121; X131; X140; X150; X151;X166; X186; X188; X205; X225; X230; X270; and X271.

In some embodiments, the polynucleotide encodes a proline hydroxylasepolypeptide capable of converting substrate compound (2) to productcompound (1) with improved properties as compared to SEQ ID NO:2,wherein the polypeptide comprises an amino acid sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to reference sequence SEQ ID NO:2,and at least a combination of residue differences as compared to SEQ IDNO: 2 selected from: (a) X103L and X166Q; (b) X52P and X255Y; (c)X4E/L/S and X115A; (d) X25R and X58A; (e) X29A and X166T/Q/L; (f)X115H/D/G and X121F; (g) X3S, X103L, and X166Q; (h) X103L, X131Y/F, andX166T/Q/L; (i) X26T, X103L and X166T/Q/L; (j) X25R, X66Q, X92V andX115E; (k) X25R, X66Q, X92V, X103L, X115E, and X166Q; and (l) X3S, X25R,X66Q, X92V, X103L, X115E, and X166Q.

In some embodiments, the polynucleotide encodes an engineered prolinehydroxylase polypeptide capable of converting substrate compound (2) toproduct compound (1) with improved enzyme properties as compared to thereference polypeptide of SEQ ID NO: 2, wherein the polypeptide comprisesan amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to areference polypeptide selected from any one of SEQ ID NO: 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and 228,with the proviso that the amino acid sequence comprises any one of theset of residue differences as compared to SEQ ID NO: 2 contained in anyone of the polypeptide sequences of SEQ ID NO: 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and 228, as listed inTables 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H.

In some embodiments, the polynucleotide encoding the engineered prolinehydroxylase comprises an polynucleotide sequence selected from SEQ IDNO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, and 227.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, and 227, or a complement thereof, and encodes apolypeptide having proline hydroxylase activity with one or more of theimproved properties described herein. In some embodiments, thepolynucleotide capable of hybridizing under highly stringent conditionsencodes a proline hydroxylase polypeptide that has an amino acidsequence comprising one or more residue differences as compared to SEQID NO: 2 at residue positions selected from: X2; X3; X4; X5; X9; X13;X17; X24; X25; X26; X29; X30; X36; X42; X52; X57; X58; X59; X62; X66;X86; X88; X92; X95; X98; X103; X112; X113; X114; X115; X116; X121; X131;X140; X150; X151; X166; X186; X188; X205; X225; X230; X270; and X271. Insome embodiments, the residue differences at residue positions X2; X3;X4; X5; X9; X13; X17; X24; X25; X26; X29; X30; X36; X42; X52; X57; X58;X59; X62; X66; X86; X88; X92; X95; X98; X103; X112; X113; X114; X115;X116; X121; X131; X140; X150; X151; X166; X186; X188; X205; X225; X230;X270; and X271 are selected from X2K; X2T; X3S; X4Q; X4L; X4E; X4S; X5I;X5L; X5M; X9I; X13T; X17V, X24R; X24S; X25R; X26R; X26T; X26W; X29A;X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G; X62Q; X66Q; X86S;X88R; X92V; X95M; X98F; X98T; X103L; X103Q; X112T; X112V; X113E; X114N;X115E; X115H; X115D; X115G; X115S; X115A; X116L; X121F; X131Y; X131F;X140L; X150S; X151A; X151H; X151S; X166T; X166L; X166Q; X186G; X188G;X205V; X225L; X225Y; X225W; X230V; X270E; X271K; and X271R.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequenceidentity at the nucleotide level to a reference polynucleotide encodingthe engineered proline hydroxylase. In some embodiments, the referencepolynucleotide sequence is selected from SEQ ID NO: 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, and 227.

An isolated polynucleotide encoding an improved proline hydroxylasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. In some embodiments, the polynucleotidesencoding the polypeptides can be provided as expression vectors whereone or more control sequences is present to regulate the expression ofthe polynucleotides and/or polypeptides. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3^(rd)Ed., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

In some embodiments, the control sequence includes among others, apromoter, leader sequence, polyadenylation sequence, propeptidesequence, signal peptide sequence, and transcription terminator.Suitable promoters can be selected based on the host cells used. Forbacterial host cells, suitable promoters for directing transcription ofthe nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. NatlAcad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer etal., 1983, Proc. Natl Acad. Sci. USA 80: 21-25). Exemplary promoters forfilamentous fungal host cells, include promoters obtained from the genesfor Aspergillus oryzae TAKA amylase, Rhizomucor miehei asparticproteinase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-likeprotease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of thepromoters from the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase), and mutant, truncated,and hybrid promoters thereof. Exemplary yeast cell promoters can be fromthe genes can be from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomycescerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention. For example, exemplary transcription terminatorsfor bacterial cells are described in Ermolaeva et al., 2001, J. Mol.Biol. 301:27-33. Exemplary transcription terminators for filamentousfungal host cells can be obtained from the genes for Aspergillus oryzaeTAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease. Exemplary terminators for yeast hostcells can be obtained from the genes for Saccharomyces cerevisiaeenolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomycescerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other usefulterminators for yeast host cells are described by Romanos et al., 1992,supra.

The control sequence may also be a suitable leader sequence thatcontains a translation initiation sequence. The leader sequence isoperably linked to the 5′ terminus of the nucleic acid sequence encodingthe polypeptide. Any leader sequence that is functional in initiatingtranslation in the host cell of choice may be used. Exemplary bacterialtranslation initiation sequence can be obtained from any expressedbacterial gene, such as from E. coli., Bacillus subtilis, Lactococcuslactic, and Sinorhizobium meliloti (see, e.g., Sakai, et al., 2001, J.Mol. Evol. 52:164-170; Ma et al., 2002, J Bacteriol. 184(20):5733-5745). In some embodiments, artificial translation initiationsequences (e.g., Shine-Delgarno sequence) can be used (see, e.g.,Vimberg et al., 2007, BMC Molecular Biology 8:100). Exemplary leadersfor filamentous fungal host cells are obtained from the genes forAspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. Any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention. Effectivesignal peptide coding regions for bacterial host cells are the signalpeptide coding regions obtained from the genes for Bacillus NC1B 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are described by Simonenand Palva, 1993, Microbiol Rev 57: 109-137. Effective signal peptidecoding regions for filamentous fungal host cells can be the signalpeptide coding regions obtained from the genes for Aspergillus oryzaeTAKA amylase, Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolenscellulase, and Humicola lanuginosa lipase. Useful signal peptides foryeast host cells can be from the genes for Saccharomyces cerevisiaealpha-factor and Saccharomyces cerevisiae invertase. Other useful signalpeptide coding regions are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila lactase (WO 95/33836). Whereboth signal peptide and propeptide regions are present at the aminoterminus of a polypeptide, the propeptide region is positioned next tothe amino terminus of a polypeptide and the signal peptide region ispositioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

In another aspect, the present disclosure is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered proline hydroxylase polypeptide, and one or more expressionregulating regions such as a promoter and a terminator, a replicationorigin, etc., depending on the type of hosts into which they are to beintroduced. The various nucleic acid and control sequences describedabove may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding thepolypeptide at such sites. Alternatively, the nucleic acid sequence ofthe present disclosure may be expressed by inserting the nucleic acidsequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector preferably contains one or more selectablemarkers, which permit easy selection of transformed cells. A selectablemarker is a gene the product of which provides for biocide or viralresistance, resistance to heavy metals, prototrophy to auxotrophs, andthe like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers, whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol (Example 1) or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved proline hydroxylasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe proline hydroxylase enzyme in the host cell. Host cells for use inexpressing the polypeptides encoded by the expression vectors are wellknown in the art and include but are not limited to, bacterial cells,such as E. coli, Bacillus subtilis, Streptomyces and Salmonellatyphimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; andplant cells. Exemplary host cells are Escherichia coli W3110 (ΔfhuA) andBL21.

Accordingly, in another aspect, the present disclosure provides methodsof manufacturing the engineered proline hydroxylase polypeptides, wherethe method can comprise culturing a host cell capable of expressing apolynucleotide encoding the proline hydroxylase polypeptide underconditions suitable for expression of the polypeptide. The method canfurther comprise isolating or purifying the expressed prolinehydroxylase polypeptide, as described herein

Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art. Polynucleotidesfor expression of the proline hydroxylase may be introduced into cellsby various methods known in the art. Techniques include among others,electroporation, biolistic particle bombardment, liposome mediatedtransfection, calcium chloride transfection, and protoplast fusion.

In the embodiments herein, the improved polypeptides and correspondingpolynucleotides can be obtained using methods used by those skilled inthe art. The parental, naturally occurring polynucleotide sequenceencoding the naturally occurring cis-4-proline hydroxylase ofSinorhizobium meliloti is described in US patent publication no.US20110091942 and International patent publication no. WO2009139365,incorporated herein by reference. The engineered proline hydroxylasesdescribed herein can be obtained by subjecting the polynucleotideencoding the naturally occurring or another engineered prolinehydroxylase to mutagenesis and/or directed evolution methods, asdiscussed herein. An exemplary directed evolution technique ismutagenesis and/or DNA shuffling as described in Stemmer, 1994, ProcNatl Acad Sci USA 91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746. Otherdirected evolution procedures that can be used include, among others,staggered extension process (StEP), in vitro recombination (Zhao et al.,1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al.,1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis (Black etal., 1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesis anddirected evolution techniques useful for the purposes herein are alsodescribed in the following references: Ling, et al., 1997, Anal.Biochem. 254(2):157-78; Dale et al., 1996, “Oligonucleotide-directedrandom mutagenesis using the phosphorothioate method,” In Methods Mol.Biol. 57:369-74; Smith, 1985, Ann. Rev. Genet. 19:423-462; Botstein etal., 1985, Science 229:1193-1201; Carter, 1986, Biochem. J. 237:1-7;Kramer et al., 1984, Cell, 38:879-887; Wells et al., 1985, Gene34:315-323; Minshull et al., 1999, Curr Opin Chem Biol 3:284-290;Christians et al., 1999, Nature Biotech 17:259-264; Crameri et al.,1998, Nature 391:288-291; Crameri et al., 1997, Nature Biotech15:436-438; Zhang et al., 1997, Proc Natl Acad Sci USA 94:45-4-4509;Crameri et al., 1996, Nature Biotech 14:315-319; Stemmer, 1994, Nature370:389-391; Stemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; WO95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767and U.S. Pat. No. 6,537,746. All publications are incorporated herein byreference.

The clones obtained following mutagenesis treatment can be screened forengineered proline hydroxylases having one or more desired improvedenzyme properties. For example, where the improved enzyme propertydesired is regioselectivity, enzyme activity may be measured forproduction of compound (1) and compound (1a). Clones containing apolynucleotide encoding a proline hydroxylase with the desiredcharacteristics, e.g., increased ratio of compound (1) over compound(1a), are then isolated, sequenced to identify the nucleotide sequencechanges (if any), and used to express the enzyme in a host cell.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry techniques, such as HPLC analysis and/orderivatization of products (pre or post separation), e.g., with dansylchloride or OPA (see, e.g., Yaegaki et al., 1986, J Chromatogr.356(1):163-70).

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides encodingportions of the proline hydroxylase can be prepared by chemicalsynthesis using, e.g., the classical phosphoramidite method described byBeaucage et al., 1981, Tet Lett 22:1859-69, or the method described byMatthes et al., 1984, EMBO J. 3:801-05, e.g., as it is typicallypracticed in automated synthetic methods. According to thephosphoramidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors. In addition, essentially any nucleic acid can beobtained from any of a variety of commercial sources. In someembodiments, additional variations can be created by synthesizingoligonucleotides containing deletions, insertions, and/or substitutions,and combining the oligonucleotides in various permutations to createengineered proline hydroxylases with improved properties.

Accordingly, in some embodiments, a method for preparing the engineeredproline hydroxylases polypeptide can comprise: (a) synthesizing apolynucleotide encoding a polypeptide comprising an amino acid sequenceselected from SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, and 228, and having one or more residuedifferences as compared to SEQ ID NO:2 at residue positions selectedfrom: X2; X3; X4; X5; X9; X13; X17; X24; X25; X26; X29; X30; X36; X42;X52; X57; X58; X59; X62; X66; X86; X88; X92; X95; X98; X103; X112; X113;X114; X115; X116; X121; X131; X140; X150; X151; X166; X186; X188; X205;X225; X230; X270; and X271; and (b) expressing the proline hydroxylasepolypeptide encoded by the polynucleotide.

In some embodiments of the method, the residue differences at residuepositions X2; X3; X4; X5; X9; X13; X17; X24; X25; X26; X29; X30; X36;X42; X52; X57; X58; X59; X62; X66; X86; X88; X92; X95; X98; X103; X112;X113; X114; X115; X116; X121; X131; X140; X150; X151; X166; X186; X188;X205; X225; X230; X270; and X271 are selected from X2K; X2T; X3S; X4Q;X4L; X4E; X4S; X5I; X5L; X5M; X9I; X13T; X17V, X24R; X24S; X25R; X26R;X26T; X26W; X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G;X62Q; X66Q; X86S; X88R; X92V; X95M; X98F; X98T; X103L; X103Q; X112T;X112V; X113E; X114N; X115E; X115H; X115D; X115G; X1155; X115A; X116L;X121F; X131Y; X131F; X140L; X1505; X151A; X151H; X1515; X166T; X166L;X166Q; X186G; X188G; X205V; X225L; X225Y; X225W; X230V; X270E; X271K;and X271R.

In some embodiments of the method, the polynucleotide can encode anengineered proline hydroxylase that has optionally one or several (e.g.,up to 3, 4, 5, or up to 10) amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the amino acid sequence hasoptionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1- 45, or 1-50 aminoacid residue deletions, insertions and/or substitutions. In someembodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

In some embodiments, any of the engineered proline hydroxylase enzymesexpressed in a host cell can be recovered from the cells and/or theculture medium using any one or more of the well known techniques forprotein purification, including, among others, lysozyme treatment,sonication, filtration, salting-out, ultra-centrifugation, andchromatography. Suitable solutions for lysing and the high efficiencyextraction of proteins from bacteria, such as E. coli, are commerciallyavailable, such as CelLytic B™ from Sigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the proline hydroxylasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved proline hydroxylase enzymes. For affinity chromatographypurification, any antibody which specifically binds the prolinehydroxylase polypeptide may be used. For the production of antibodies,various host animals, including but not limited to rabbits, mice, rats,etc., may be immunized by injection with a proline hydroxylasepolypeptide, or a fragment thereof. The proline hydroxylase polypeptideor fragment may be attached to a suitable carrier, such as BSA, by meansof a side chain functional group or linkers attached to a side chainfunctional group. In some embodiments, the affinity purification can usea specific ligand bound by the proline hydroxylase, such aspoly(L-proline) or dye affinity column (see, e.g., EP0641862;Stellwagen, E., 2001, “Dye Affinity Chromatography,” In CurrentProtocols in Protein Science Unit 9.2-9.2.16).

5.7 Methods of Using the Engineered Proline Hydroxylase Enzymes

In another aspect, the proline hydroxylases described herein can be usedin a process for converting a suitable substrate to its hydroxylatedproduct. Generally, the process for performing the hydroxylationreaction comprises contacting or incubating the substrate compound inpresence of a co-substrate, such as α-ketoglutarate, with a prolinehydroxylase polypeptide of the disclosure under reaction conditionssuitable for formation of the hydroxylated product.

In some embodiments, the proline hydroxylases can be used in theconversion of substrate compound (II) to product compound (I), asillustrated in Scheme 2:

wherein

L is selected from the group consisting of a bond, (C₁-C₄)alkylene and(C₂-C₄)alkenylene;

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² and R³ are each independently selected from the group consisting ofhydrogen and optionally substituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and(C₂-C₆)alkynyl;

R⁴ is selected from the group consisting of optionally substituted(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, aryl, heteroaryl,cycloalkyl, heterocycloalkyl; or R⁴ together with one of R¹ or R² is a(C₁-C₅)alkylene or (C₂-C₅)alkenylene and forms a 5- to 8-memberedheterocyclic ring containing the nitrogen atom, wherein the ring isoptionally substituted with 1 to 4 independently selected R⁶ groups;

R⁵ is hydrogen or a bond that forms an epoxide with a carbon atom of L;

each occurrence of R⁶ is independently selected from the groupconsisting of halo, (C₁-C₆)alkyl, and (C₁-C₆)alkyloxy; and

----- represents an optional bond to a carbon atom of L to form a doublebond,

with the provisos that

(i) when R⁴ does not form a ring with one R² or R³, or when R⁴ forms a5-membered heterocyclic ring containing the nitrogen atom with one of R²or R³, then L is a methylene;

(ii) when R⁴ forms a 6-membered heterocyclic ring containing thenitrogen atom with one of R² or R³, then L is a bond or ethylene; and

(iii) when R⁵ is a bond to a carbon atom of L to form an epoxide, thenR⁴ forms the heterocyclic ring containing the nitrogen atom with one ofR² or R³ and L is a (C₁-C₄)alkylene or (C₂-C₄)alkenylene.

Accordingly, in some embodiments, a process for preparing productcompound (I) can comprise contacting the substrate compound of formula(II)

wherein

L, R¹, R², R³, R⁴, and R⁶ are as defined above,

----- represents an optional bond to a carbon atom of L to form a doublebond

with an engineered polypeptide disclosed herein in presence of aco-substrate under suitable reaction conditions.

In some embodiments of the process, the compound of formula (I)comprises the compound of formula (Ia),

wherein

Q is selected from the group consisting of a (C₁-C₅)alkylene and(C₂-C₅)alkenylene;

L is selected from the group consisting of a bond, (C₁-C₄)alkylene and(C₂-C₄)alkenylene;

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² is selected from the group consisting of hydrogen and optionallysubstituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and (C₂-C₆)alkynyl;

R⁵ is hydrogen, or a bond to a carbon atom of L to form an epoxide;

each occurrence of R⁶ is selected from the group consisting of halo,(C₁-C₆)alkyl, and (C₁-C₆)alkyloxy; and

q is an integer from 0 to 4;

wherein the sum of ring carbon atoms for Q+L is an integer from 2 to 5;

with the provisos that

(i) when the sum of ring carbon atoms for Q+L is 2, then L is amethylene; and

(ii) when the sum of ring carbon atoms for Q+L is 3, then L is either abond or ethylene.

Accordingly, a process for preparing the compound of formula (Ia)comprises contacting the compound of formula (IIa),

wherein

L, Q, R¹, R², R⁶, and q are as defined above for the compound of formula(Ia); and

----- represents an optional bond to a carbon atom of L to form a doublebond;

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions.

In some embodiments of the process, the compound of formula (Ia)comprises the compound of formula (Ib),

wherein

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² is selected from the group consisting of hydrogen and optionallysubstituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and (C₂-C₆)alkynyl;

each occurrence of R⁶ is independently selected from the groupconsisting of halo, (C₁-C₆)alkyl, and (C₁-C₆)alkyloxy; and

k is an integer from 1 to 5;

r is an integer from 0 to 4;

wherein k+r is 3, 4 or 5; and

q is an integer from 0 to 4;

with the proviso that when k+r is 3, then k is 1 or 3.

Accordingly, a process for preparing the compound of formula (Ib)comprises contacting the compound of formula (IIb),

wherein

R¹, R², R⁶, k, r and q are as defined above for the compound of formula(Ib);

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions. In some embodiments, kis 1 and r is 2, 3, or 4.

In some embodiments of the process, the compound of formula (Ia)comprises the compound of formula (Ic),

wherein

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² is selected from the group consisting of hydrogen and optionallysubstituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and (C₂-C₆)alkynyl;

each occurrence of R⁶ is independently selected from the groupconsisting of hydrogen, halo, (C₁-C₆)alkyl, and (C₁-C₆)alkyloxy; and

q is an integer from 0 to 4.

Accordingly, a process for preparing the compound of formula (Ic)comprises contacting the compound of formula (IIc),

wherein

R¹, R², R⁶ and q are as defined above for the compound of formula (Ic);

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions.

In some embodiments of the process, the compound of formula (Ic) isformed in excess of product compound of formula (Ic3),

Accordingly, a process for preparing the compound of formula (Ic) inexcess of the compound of formula (Ic3) comprises contacting thecompound of formula (IIc) with an engineered polypeptide describedherein having regioselectivity for product compound (1) over productcompound (1a) in presence of a co-substrate under suitable reactionconditions. In some embodiments of the process, the product compound offormula (Ic) is formed in excess of the product compound of formula(Ic3), where the ratio of compound (Ic) formed over compound (Ic3) is atleast 1.5, 2, 3, 4, 5, or 6 or greater.

In some embodiments of the process, product compound (Ic) is formed indiastereomeric excess of compound (IcR),

In some embodiments of the process, the product compound (Ic) is formedin at least 90%, 95%, 96%, 97%, 98%, 99%, or greater diastereomericexcess of compound (IcR). In some embodiments, no detectable amount ofcompound (IcR) is formed in the process.

In some embodiments of the process for preparation of the productcompounds of formula (Ic), R′ is hydroxy, R² is hydrogen, and q is 0. Assuch, in some embodiments of the process, the compound of formula (I)comprises the compound of formula (1),

Accordingly, a process for preparing the compound of formula (1)comprises contacting the compound of formula (2),

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions.

In some embodiments of the process, the product compound (1) is formedin excess of product compound (1a). In some embodiments, the productcompound (1) is formed in excess over compound (1a) in a ratio of atleast 1.5, 2, 3, 4, 5 or 6 or greater. In some embodiments of theprocess, the engineered polypeptide useful for preparing compound (1) inexcess of the compound of formula (1a) comprises contacting the compoundof formula (2) with an engineered polypeptide described herein havingregioselectivity for product compound (1) over compound (1a) undersuitable reaction conditions.

In some embodiments of the process, product compound (1) is formed indiastereomeric excess of compound (1R),

In some embodiments of the process, the product compound (1) is formedin at least 90%, 95%, 96%, 97%, 98%, 99%, or greater diastereomericexcess of compound (1R). In some embodiments, no detectable amount ofcompound (1R) is formed in the process.

In some embodiments, the compound of formula (Ia) comprises the compoundof formula (Ie),

wherein

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² is selected from the group consisting of hydrogen and optionallysubstituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and (C₂-C₆)alkynyl;

each occurrence of R⁶ is independently selected from the groupconsisting of hydrogen, halo, (C₁-C₆)alkyl, and (C₁-C₆)alkyloxy; and

q is an integer from 0 to 3.

Accordingly, in some embodiments, a process for preparing the productcompound of formula (Ie) comprises contacting the compound of formula(IIe),

wherein R¹, R², R⁶ and q are as defined for the compound of formula(Ie),

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions.

In some embodiments of the process, the compound of formula (Ie) isformed in diastereomeric excess of compound of formula (IeR),

In some embodiments of the process, the product compound of formula (Ie)is formed in at least 90%, 95%, 96%, 97%, 98%, 99%, or greaterdiastereomeric excess of the compound of formula (IeR). In someembodiments, no detectable amount of the compound of formula (IeR) isformed.

In some embodiments of the process for preparing the product compound offormula (Ie), R¹ is hydroxy, R² is hydrogen, and q is 0. As such, insome embodiments, the compound of formula (Ie) comprises compound (3),

wherein the process for preparing compound (3) comprises contactingcompound (4),

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions.

In some embodiments of the process, compound (3) is formed indiastereomeric excess of compound (3R),

In some embodiments of the process, the product compound (3) is formedin at least 90%, 95%, 96%, 97%, 98%, 99%, or greater diastereomericexcess of the compound (3R). In some embodiments, no detectable amountof the compound (3R) is formed.

In some embodiments, the compound of formula (Ia) comprises compound(5),

Accordingly, a process for preparing compound (5) comprises contactingcompound (6);

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions.

In some embodiments, the compound of formula (I) comprises the compoundof formula (III),

wherein,

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² and R³ are each independently selected from the group consisting ofhydrogen and optionally substituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and(C₂-C₆)alkynyl; and

R⁴ is selected from the group consisting of optionally substitutedalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, andheterocycloalkyl;

In some embodiments, the optionally substituted alkyl is selected fromthe group consisting of carboxy(C₁-C₆)alkyl, aminocarbonyl(C₁-C₆)alkyl,amino(C₁-C₆)alkyl, thiol(C₁-C₆)alkyl, and alkylthio(C₁-C₆)alkyl,alkylsulfonyl(C₁-C₆)alkyl, aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,cycloalkyl(C₁-C₆)alkyl, and heterocycloalkyl(C₁-C₆)alkyl.

Accordingly, in some embodiments, a process for preparing the compoundof formula (III) comprises contacting the compound of formula (IV),

wherein,

R¹, R², R³ and R⁴ are as defined above for the compound of formula(III),

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions.

In some embodiments, the engineered polypeptides can be used in aprocess for carrying out the conversion shown in Scheme 3,

wherein,

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² and R³ are independently selected from the group consisting ofhydrogen and optionally substituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and(C₂-C₆)alkynyl;

each occurrence of R⁶ is independently selected from the groupconsisting of halo, (C₁-C₆)alkyl, and (C₁-C₆)alkyloxy;

R² is selected from the group consisting of hydrogen, halo, andoptionally substituted (C₁-C₆)alkyl and (C₁-C₆)alkyloxy; or R² togetherwith one of R² or R³ forms a 5- to 7-membered heterocyclic ringcontaining the nitrogen atom;

q is an integer from 0 to 4; and

----- represent optional double bonds that form an aryl ring.

Accordingly, in some embodiments, a process for preparing the compoundof formula (V) comprises contacting the compound of formula (VI),

wherein R¹, R², R³, R⁶, R⁷, and q are as defined for the compound offormula (V),

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions.

In some embodiments, the compound of formula (V) comprises the compoundof formula (Va),

wherein,

R¹ is selected from the group consisting of hydroxy, amino,(C₁-C₆)alkyloxy, aryloxy, (C₁-C₆)alkylthio and arylthio;

R² and R³ are independently selected from the group consisting ofhydrogen and optionally substituted (C₁-C₆)alkyl, (C₂-C₆)alkenyl, and(C₂-C₆)alkynyl;

each occurrence of R⁶ is independently selected from the groupconsisting of halo, (C₁-C₆)alkyl, and (C₁-C₆)alkyloxy; and

q is an integer from 0 to 4.

Accordingly, in some embodiments, a process for preparing the compoundof formula (Va) comprises contacting the compound of formula (VIa),

wherein R¹, R², R³, R⁶, and q are as defined above for the compound offormula (Va),

with an engineered polypeptide of the disclosure in presence of aco-substrate under suitable reaction conditions.

For the foregoing processes, any of the engineered proline hydroxylasesdescribed herein can be used. By way of example and without limitation,in some embodiments, the processes can use an engineered prolinehydroxylase polypeptide comprising an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identity to a reference sequence selected from SEQID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, and 228.

In some embodiments of the processes, the engineered proline hydroxylasepolypeptide comprises an amino acid sequence having one or more residuedifferences as compared to SEQ ID NO:2 at residue positions X2; X3; X4;X5; X9; X13; X17; X24; X25; X26; X29; X30; X36; X42; X52; X57; X58; X59;X62; X66; X86; X88; X92; X95; X98; X103; X112; X113; X114; X115; X116;X121; X131; X140; X150; X151; X166; X186; X188; X205; X225; X230; X270;and X271.

In some embodiments of the processes, the residue differences at residuepositions X2; X3; X4; X5; X9; X13; X17; X24; X25; X26; X29; X30; X36;X42; X52; X57; X58; X59; X62; X66; X86; X88; X92; X95; X98; X103; X112;X113; X114; X115; X116; X121; X131; X140; X150; X151; X166; X186; X188;X205; X225; X230; X270; and X271 are selected from X2K; X2T; X3S; X4Q;X4L; X4E; X4S; X5I; X5L; X5M; X9I; X13T; X17V, X24R; X24S; X25R; X26R;X26T; X26W; X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G;X62Q; X66Q; X86S; X88R; X92V; X95M; X98F; X98T; X103L; X103Q; X112T;X112V; X113E; X114N; X115E; X115H; X115D; X115G; X115S; X115A; X116L;X121F; X131Y; X131F; X140L; X150S; X151A; X151H; X151S; X166T; X166L;X166Q; X186G; X188G; X205V; X225L; X225Y; X225W; X230V; X270E; X271K;and X271R.

As noted above, in some embodiments for preparing product compound offormula (Ic) in excess over the compound of formula (Ic3), or forpreparing compound (1) in excess over compound (1a), the engineeredpolypeptide can comprise an amino acid sequence having one or morefeatures selected from: X103L; X115E; X131Y and X166Q. Exemplaryengineered polypeptides with the relevant regioselectivity can comprisean amino acid sequence selected from the group consisting of SEQ ID NO:10, 24, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, and 228.

In some embodiments, exemplary proline hydroxylases polypeptides capableof carrying out the processes herein can have an amino acid sequencecomprising a sequence selected from SEQ ID NO: 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and 228. Guidance onthe choice and use of the engineered proline hydroxylases is provided inthe descriptions herein, for example Tables 2A, 2B, 2C, 2D, 2E, 2F, 2G,and 2H and the Examples.

In the embodiments herein and illustrated in the Examples, variousranges of suitable reaction conditions that can be used in theprocesses, include but are not limited to, substrate loading,co-substrate loading, reductant, divalent transition metal, pH,temperature, buffer, solvent system, polypeptide loading, and reactiontime. Further suitable reaction conditions for carrying out the processfor biocatalytic conversion of substrate compounds to product compoundsusing an engineered proline hydroxylase polypeptide described herein canbe readily optimized in view of the guidance provided herein by routineexperimentation that includes, but is not limited to, contacting theengineered proline hydroxylase polypeptide and substrate compound underexperimental reaction conditions of concentration, pH, temperature, andsolvent conditions, and detecting the product compound.

Suitable reaction conditions using the engineered proline hydroxylasepolypeptides typically comprise a co-substrate, which is usedstoichiometrically in the hydroxylation reaction. Generally, theco-substrate for proline hydroxylases is α-ketoglutarate, also referredto as α-ketoglutaric acid and 2-oxoglutaric acid. Other analogs ofα-ketoglutarate that are capable of serving as co-substrates for prolinehydroxylases can be used. An exemplary analog that may serve as aco-substrate is 2-oxoadipate. Because the co-substrate is usedstoichiometrically, the co-substrate is present at an equimolar orhigher amount than that of the substrate compound, i.e., the molarconcentration of co-substrate is equivalent to or higher than the molarconcentration of substrate compound. In some embodiments, the suitablereaction conditions can comprise a co-substrate molar concentration ofat least 1 fold, 1.5 fold, 2 fold, 3 fold 4 fold or 5 fold or more thanthe molar concentration of the substrate compound. In some embodiments,the suitable reaction conditions can comprise a co-substrateconcentration, particularly α-ketoglutarate, of about 0.001 M to about 2M, 0.01 M to about 2 M, 0.1 M to about 2 M, 0.2 M to about 2 M, about0.5 M to about 2 M, or about 1 M to about 2 M. In some embodiments, thereaction conditions comprise a co-substrate concentration of about0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.5, or 2 M. Insome embodiments, additional co-substrate can be added during thereaction.

Substrate compound in the reaction mixtures can be varied, taking intoconsideration, for example, the desired amount of product compound, theeffect of substrate concentration on enzyme activity, stability ofenzyme under reaction conditions, and the percent conversion ofsubstrate to product. In some embodiments, the suitable reactionconditions comprise a substrate compound loading of at least about 0.5to about 200 g/L, 1 to about 200 g/L, 5 to about 150 g/L, about 10 toabout 100 g/L, 20 to about 100 g/L or about 50 to about 100 g/L. In someembodiments, the suitable reaction conditions comprise a substratecompound loading of at least about 0.5 g/L, at least about 1 g/L, atleast about 5 g/L, at least about 10 g/L, at least about 15 g/L, atleast about 20 g/L, at least about 30 g/L, at least about 50 g/L, atleast about 75 g/L, at least about 100 g/L, at least about 150 g/L or atleast about 200 g/L, or even greater. The values for substrate loadingsprovided herein are based on the molecular weight of compound (2),however it also contemplated that the equivalent molar amounts ofvarious hydrates and salts of compound (2) also can be used in theprocess. In addition, substrate compounds covered by formulas (II) and(VI), including compounds of formula (IIa), (IVa) and (VIa) can also beused in appropriate amounts, in light of the amounts used for compound(2).

In carrying out the proline hydroxylase mediated processes describedherein, the engineered polypeptide may be added to the reaction mixturein the form of a purified enzyme, partially purified enzyme, whole cellstransformed with gene(s) encoding the enzyme, as cell extracts and/orlysates of such cells, and/or as an enzyme immobilized on a solidsupport. Whole cells transformed with gene(s) encoding the engineeredproline hydroxylase enzyme or cell extracts, lysates thereof, andisolated enzymes may be employed in a variety of different forms,including solid (e.g., lyophilized, spray-dried, and the like) orsemisolid (e.g., a crude paste). The cell extracts or cell lysates maybe partially purified by precipitation (ammonium sulfate,polyethyleneimine, heat treatment or the like, followed by a desaltingprocedure prior to lyophilization (e.g., ultrafiltration, dialysis, andthe like). Any of the enzyme preparations (including whole cellpreparations) may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde or immobilization to asolid phase (e.g., Eupergit C, and the like).

The gene(s) encoding the engineered proline hydroxylase polypeptides canbe transformed into host cell separately or together into the same hostcell. For example, in some embodiments one set of host cells can betransformed with gene(s) encoding one engineered proline hydroxylasepolypeptide and another set can be transformed with gene(s) encodinganother engineered proline hydroxylase polypeptide. Both sets oftransformed cells can be utilized together in the reaction mixture inthe form of whole cells, or in the form of lysates or extracts derivedtherefrom. In other embodiments, a host cell can be transformed withgene(s) encoding multiple engineered proline hydroxylase polypeptide. Insome embodiments the engineered polypeptides can be expressed in theform of secreted polypeptides and the culture medium containing thesecreted polypeptides can be used for the proline hydroxylase reaction.

The improved activity and/or stereoselectivity of the engineered prolinehydroxylase polypeptides disclosed herein provides for processes whereinhigher percentage conversion can be achieved with lower concentrationsof the engineered polypeptide. In some embodiments of the process, thesuitable reaction conditions comprise an engineered polypeptide amountof about 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 20% (w/w), 30% (w/w),40% (w/w), 50% (w/w), 75% (w/w), 100% (w/w) or more of substratecompound loading.

In some embodiments, the engineered polypeptide is present at about 0.01g/L to about 50 g/L; about 0.05 g/L to about 50 g/L; about 0.1 g/L toabout 40 g/L; about 1 g/L to about 40 g/L; about 2 g/L to about 40 g/L;about 5 g/L to about 40 g/L; about 5 g/L to about 30 g/L; about 0.1 g/Lto about 10 g/L; about 0.5 g/L to about 10 g/L; about 1 g/L to about 10g/L; about 0.1 g/L to about 5 g/L; about 0.5 g/L to about 5 g/L; orabout 0.1 g/L to about 2 g/L. In some embodiments, the prolinehydroxylase polypeptide is present at about 0.01 g/L, 0.05 g/L, 0.1 g/L,0.2 g/L, 0.5 g/L, 1, 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30g/L, 35 g/L, 40 g/L, or 50 g/L.

In some embodiments, the reactions conditions also comprise a divalenttransition metal capable of serving as a cofactor in the oxidationreaction. Generally, the divalent transition metal co-factor is ferrousion, i.e., Fe′. The ferrous ion may be provided in various forms, suchas ferrous sulfate (FeSO₄), ferrous chloride (FeCl₂), ferrous carbonate(FeCO₃), and the salts of organic acids such as citrates, lactates andfumarates. An exemplary source of ferrous sulfate is Mohr's salt, whichis ferrous ammonium sulfate (NH₄)₂Fe(SO₄)₂ and is available in anhydrousand hydrated (i.e., hexahydrate) forms. While ferrous ion is thetransition metal co-factor found in the naturally occurring prolinehydroxylase and functions efficiently in the engineered enzymes, it isto be understood that other divalent transition metals capable of actingas a co-factor can be used in the processes. In some embodiments, thedivalent transition metal co-factor can comprise Mn⁺² and Cr⁺². In someembodiments, the reaction conditions can comprises a divalent transitionmetal cofactor, particularly Fe⁺², at a concentration of about 0.1 mM to10 mM, 0.1 mM to about 5 mM, 0.5 mM to about 5 mM, about 0.5 mM to about3 mM or about 1 mM to about 2 mM. In some embodiments, the reactionconditions comprise a divalent transition metal co-factor concentrationof about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 5 mM, 7.5 mMor 10 mM. In some embodiments, higher concentrations of divalenttransition metal cofactor can be used, for example up to 50 mM or up to100 mM.

In some embodiments, the reaction conditions can further comprise areductant capable of reducing ferric ion, Fe⁺³ to ferrous ion, Fe⁺². Insome embodiments, the reductant comprises ascorbic acid, typicallyL-ascorbic acid. While ascorbic acid is not required for thehydroxylation reaction, enzymatic activity is enhanced in its presence.Without being bound by theory, the ascorbate is believed to maintain orregenerate the enzyme-Fe⁺² form, which is the active form mediating thehydroxylation reaction. Generally, the reaction conditions can comprisean ascorbic acid concentration that corresponds proportionately to thesubstrate loading. In some embodiments, the ascorbic acid is present inat least about 0.1 fold, 0.2 fold 0.3 fold, 0.5 fold, 0.75 fold, 1 fold,1.5 fold, or at least 2 fold the molar amount of substrate. In someembodiments, the reductant, particularly L-ascorbic acid, is at aconcentration of about 0.001 M to about 0.5 M, about 0.01M to about 0.5M, about 0.01 M to about 0.4 M, about 0.1 to about 0.4 M, or about 0.1to about 0.3 M. In some embodiments, the reductant, particularlyascorbic acid, is at a concentration of about 0.001 M, 0.005 M, 0.01 M,0.02M, 0.03 M, 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.3 M, 0.4 M, or 0.5 M.

In some embodiments, the reaction conditions comprise molecular oxygen,i.e., O₂. Without being bound by theory, one atom of oxygen frommolecular oxygen is incorporated into the substrate compound to form thehydroxylated product compound. The O₂ may be present naturally in thereaction solution, or introduced and/or supplemented into the reactionartificially. In some embodiments, the reaction conditions can compriseforced aeration (e.g., sparging) with air, 02 gas, or other02-containing gases. In some embodiments, the O₂ in the reaction can beincreased by increasing the pressure of the reaction with O₂ or anO₂-containing gas. This can be done by carrying out the reaction in avessel that can be pressurized with 02 gas. In some embodiments, the 02gas can be sparged through the reaction solution at a rate of at least 1liter per hour (L/h), at least 2 L/h, at least 3 L/h, at least 4 L/h, atleast 5 L/h, or greater. In some embodiments, the O₂ gas can be spargedthrough the reaction solution at a rate of between about 1 L/h and 10L/h, between about 2 L/h and 7 L/h, or between about 3 L/h and 5 L/h.

During the course of the reaction, the pH of the reaction mixture maychange. The pH of the reaction mixture may be maintained at a desired pHor within a desired pH range. This may be done by the addition of anacid or a base, before and/or during the course of the reaction.Alternatively, the pH may be controlled by using a buffer. Accordingly,in some embodiments, the reaction condition comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,by way of example and not limitation, borate, phosphate,2-(N-morpholino)ethanesulfonic acid (MES),3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine,and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), and the like. Insome embodiments, the buffer is phosphate. In some embodiments of theprocess, the suitable reaction conditions comprise a buffer (e.g.,phosphate) concentration of from about 0.01 to about 0.4 M, 0.05 toabout 0.4 M, 0.1 to about 0.3 M, or about 0.1 to about 0.2 M. In someembodiments, the reaction condition comprises a buffer (e.g., phosphate)concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12,0.14, 0.16, 0.18, 0.2, 0.3, or 0.4 M. In some embodiments, the reactionconditions comprise water as a suitable solvent with no buffer present.

In the embodiments of the process, the reaction conditions can comprisea suitable pH. The desired pH or desired pH range can be maintained byuse of an acid or base, an appropriate buffer, or a combination ofbuffering and acid or base addition. The pH of the reaction mixture canbe controlled before and/or during the course of the reaction. In someembodiments, the suitable reaction conditions comprise a solution pHfrom about 4 to about 10, pH from about 5 to about 10, pH from about 5to about 9, pH from about 6 to about 9, pH from about 6 to about 8. Insome embodiments, the reaction conditions comprise a solution pH ofabout 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

In the embodiments of the processes herein, a suitable temperature canbe used for the reaction conditions, for example, taking intoconsideration the increase in reaction rate at higher temperatures, andthe activity of the enzyme during the reaction time period. Accordingly,in some embodiments, the suitable reaction conditions comprise atemperature of about 10° C. to about 60° C., about 10° C. to about 55°C., about 15° C. to about 60° C., about 20° C. to about 60° C., about20° C. to about 55° C., about 25° C. to about 55° C., or about 30° C. toabout 50° C. In some embodiments, the suitable reaction conditionscomprise a temperature of about 10° C., 15° C., 20° C., 25° C., 30° C.,35° C., 40° C., 45° C., 50° C., 55° C., or 60° C. In some embodiments,the temperature during the enzymatic reaction can be maintained at aspecific temperature throughout the course of the reaction. In someembodiments, the temperature during the enzymatic reaction can beadjusted over a temperature profile during the course of the reaction.

The processes of the disclosure are generally carried out in a solvent.Suitable solvents include water, aqueous buffer solutions, organicsolvents, polymeric solvents, and/or co-solvent systems, which generallycomprise aqueous solvents, organic solvents and/or polymeric solvents.The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. In some embodiments, the processes using theengineered proline hydroxylase polypeptides can be carried out in anaqueous co-solvent system comprising an organic solvent (e.g., ethanol,isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF)ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl t butylether (MTBE), toluene, and the like), ionic or polar solvents (e.g.,1-ethyl 4 methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl 3methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol,and the like). In some embodiments, the co-solvent can be a polarsolvent, such as a polyol, dimethylsulfoxide (DMSO), or lower alcohol.The non-aqueous co-solvent component of an aqueous co-solvent system maybe miscible with the aqueous component, providing a single liquid phase,or may be partly miscible or immiscible with the aqueous component,providing two liquid phases. Exemplary aqueous co-solvent systems cancomprise water and one or more co-solvents selected from an organicsolvent, polar solvent, and polyol solvent. In general, the co-solventcomponent of an aqueous co-solvent system is chosen such that it doesnot adversely inactivate the proline hydroxylase enzyme under thereaction conditions. Appropriate co-solvent systems can be readilyidentified by measuring the enzymatic activity of the specifiedengineered proline hydroxylase enzyme with a defined substrate ofinterest in the candidate solvent system, utilizing an enzyme activityassay, such as those described herein.

In some embodiments of the process, the suitable reaction conditionscomprise an aqueous co-solvent, where the co-solvent comprises DMSO atabout 1% to about 50% (v/v), about 1 to about 40% (v/v), about 2% toabout 40% (v/v), about 5% to about 30% (v/v), about 10% to about 30%(v/v), or about 10% to about 20% (v/v). In some embodiments of theprocess, the suitable reaction conditions can comprise an aqueousco-solvent comprising DMSO at about 1% (v/v), about 5% (v/v), about 10%(v/v), about 15% (v/v), about 20% (v/v), about 25% (v/v), about 30%(v/v), about 35% (v/v), about 40% (v/v), about 45% (v/v), or about 50%(v/v).

In some embodiments, the reaction conditions can comprise a surfactantfor stabilizing or enhancing the reaction. Surfactants can comprisenon-ionic, cationic, anionic and/or amphiphilic surfactants. Exemplarysurfactants, include by way of example and not limitation, nonylphenoxypolyethoxylethanol (NP40), Triton X-100,polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodiumoleylamidosulfate, polyoxyethylene-sorbitanmonostearate,hexadecyldimethylamine, etc. Any surfactant that may stabilize orenhance the reaction may be employed. The concentration of thesurfactant to be employed in the reaction may be generally from 0.1 to50 mg/ml, particularly from 1 to 20 mg/ml.

In some embodiments, the reaction conditions can include an antifoamagent, which aids in reducing or preventing formation of foam in thereaction solution, such as when the reaction solutions are mixed orsparged. Anti-foam agents include non-polar oils (e.g., minerals,silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alkylamides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica,polypropylene, etc.), some of which also function as surfactants.Exemplary anti-foam agents include, Y-30® (Dow Corning), poly-glycolcopolymers, oxy/ethoxylated alcohols, and polydimethylsiloxanes. In someembodiments, the anti-foam can be present at about 0.001% (v/v) to about5% (v/v), about 0.01% (v/v) to about 5% (v/v), about 0.1% (v/v) to about5% (v/v), or about 0.1% (v/v) to about 2% (v/v). In some embodiments,the anti-foam agent can be present at about 0.001% (v/v), about 0.01%(v/v), about 0.1% (v/v), about 0.5% (v/v), about 1% (v/v), about 2%(v/v), about 3% (v/v), about 4% (v/v), or about 5% (v/v) or more asdesirable to promote the reaction.

The quantities of reactants used in the hydroxylase reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of proline hydroxylase substrate employed.Those having ordinary skill in the art will readily understand how tovary these quantities to tailor them to the desired level ofproductivity and scale of production.

In some embodiments, the order of addition of reactants is not critical.The reactants may be added together at the same time to a solvent (e.g.,monophasic solvent, biphasic aqueous co-solvent system, and the like),or alternatively, some of the reactants may be added separately, andsome together at different time points. For example, the cofactor,co-substrate, proline hydroxylase, and substrate may be added first tothe solvent.

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a pre-chilled lyophilizationchamber, followed by the application of a vacuum.

For improved mixing efficiency when an aqueous co-solvent system isused, the proline hydroxylase, and cofactor may be added and mixed intothe aqueous phase first. The organic phase may then be added and mixedin, followed by addition of the proline hydroxylase substrate andco-substrate. Alternatively, the proline hydroxylase substrate may bepremixed in the organic phase, prior to addition to the aqueous phase.

The hydroxylation process is generally allowed to proceed until furtherconversion of substrate to hydroxylated product does not changesignificantly with reaction time, e.g., less than 10% of substrate beingconverted, or less than 5% of substrate being converted). In someembodiments, the reaction is allowed to proceed until there is completeor near complete conversion of substrate to product. Transformation ofsubstrate to product can be monitored using known methods by detectingsubstrate and/or product, with or without derivatization. Suitableanalytical methods include gas chromatography, HPLC, MS, and the like.

In some embodiments of the process, the suitable reaction conditionscomprise a substrate loading of at least about 5 g/L, 10 g/L, 20 g/L, 30g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, or more, and wherein themethod results in at least about 50%, 60%, 70%, 80%, 90%, 95% or greaterconversion of substrate compound to product compound in about 48h orless, in about 36 h or less, or in about 24 h or less.

The engineered proline hydroxylase polypeptides of the presentdisclosure when used in the process under suitable reaction conditionsresult in an excess of the cis-hydroxylated product in at least 90%,95%, 96%, 97%, 98%, 99%, or greater diastereomeric excess over thetrans-hydroxylated product. In some embodiments, no detectable amount ofcompound trans-hydroxylated product is formed.

In further embodiments of the processes for converting substratecompound to hydroxylated product compound using the engineered prolinehydroxylase polypeptides, the suitable reaction conditions can comprisean initial substrate loading to the reaction solution which is thencontacted by the polypeptide. This reaction solution is then furthersupplemented with additional substrate compound as a continuous orbatchwise addition over time at a rate of at least about 1 g/L/h, atleast about 2 g/L/h, at least about 4 g/L/h, at least about 6 g/L/h, orhigher. Thus, according to these suitable reaction conditions,polypeptide is added to a solution having an initial substrate loadingof at least about 20 g/L, 30 g/L, or 40 g/L. This addition ofpolypeptide is then followed by continuous addition of further substrateto the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6 g/L/h until amuch higher final substrate loading of at least about 30 g/L, 40 g/L, 50g/L, 60 g/L, 70 g/L, 100 g/L, 150 g/L, 200 g/L or more, is reached.Accordingly, in some embodiments of the process, the suitable reactionconditions comprise addition of the polypeptide to a solution having aninitial substrate loading of at least about 20 g/L, 30 g/L, or 40 g/Lfollowed by addition of further substrate to the solution at a rate ofabout 2 g/L/h, 4 g/L/h, or 6 g/L/h until a final substrate loading of atleast about 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L or more, isreached. This substrate supplementation reaction condition allows forhigher substrate loadings to be achieved while maintaining high rates ofconversion of substrate to hydroxylated product of at least about 50%,60%, 70%, 80%, 90% or greater conversion of substrate. In someembodiments of this process, the substrate added is in a solutioncomprising α-ketoglutarate at an equimolar or higher amount of thefurther added substrate.

In some embodiments of the processes, the reaction using an engineeredproline hydroxylase polypeptide can comprise the following suitablereaction conditions: (a) substrate loading at about 5 g/L to 30 g/L; (b)about 0.1 g/L to 10 g/L of the engineered polypeptide; (c) about 19 g/L(0.13 M) to 57 g/L (0.39 M) of α-ketoglutarate; (d) about 14 g/L (0.08M) to 63 g/L (0.36 M) ascorbic acid; (e) about 1.5 g/L (3.8 mM) to 4.5g/L (11.5 mM) of FeSO₄; (f) a pH of about 6 to 7; (g) temperature ofabout 20° to 40° C.; and (h) reaction time of 2-24 h.

In some embodiments of the processes, the reaction using an engineeredproline hydroxylase polypeptide can comprise the following suitablereaction conditions: (a) substrate loading at about 10 g/L to 100 g/L;(b) about 1 g/L to about 50 g/L of engineered polypeptide; (c)α-ketoglutarate at about 1 to 2 molar equivalents of substrate compound;(d) ascorbic acid at about 0.25 to 0.75 molar equivalents of substratecompound; (e) about 0.5 mM to about 12 mM of FeSO₄; (f) pH of about 6 to8; (g) temperature of about 20° to 40° C.; and (h) reaction time of 6 to120 h.

In some embodiments, additional reaction components or additionaltechniques carried out to supplement the reaction conditions. These caninclude taking measures to stabilize or prevent inactivation of theenzyme, reduce product inhibition, shift reaction equilibrium tohydroxylated product formation.

In further embodiments, any of the above described process for theconversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction; isolation;purification; and crystallization of product compound. Methods,techniques, and protocols for extracting, isolating, purifying, and/orcrystallizing the hydroxylated product from biocatalytic reactionmixtures produced by the above disclosed processes are known to theordinary artisan and/or accessed through routine experimentation.Additionally, illustrative methods are provided in the Examples below.

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

6. EXAMPLES Example 1: Synthesis, Optimization, and Screening EngineeredProline Hydroxylase Polypeptides

Gene Synthesis and Optimization:

The polynucleotide sequence encoding the reported wild-typecis-4-proline hydroxylase polypeptide from Sinorhizobium meliloti, asrepresented by SEQ ID NO: 2, was synthesized as the gene of SEQ IDNO: 1. The synthetic gene of SEQ ID NO: 1 was cloned into a pCK110900vector system (see e.g., US Patent Application Publication 20060195947,which is hereby incorporated by reference herein) and subsequentlyexpressed in E. coli W3110fhuA. The E. coli W3110 expresses the prolinehydroxylase polypeptides under the control of the lac promoter. Based onsequence comparisons with other proline hydroxylases and computermodeling of the enzyme structure docked to the substrate proline,residue positions associated with the active site, peptide loops,solution/substrate interface, and potential stability positions wereidentified and subjected to mutagenesis. These first round variants werescreened under HTP Assay conditions with (2S)-piperidine-2-carboxylicacid as substrate. Variants with increased enzymatic activity and/orexpression were identified. Two additional codon optimizedpolynucleotides encoding the amino acid sequence of the naturallyoccurring enzyme was also generated (i.e., SEQ ID NO:3 and 5) forcomparison purposes. The codon optimized polynucleotides 3 and 5expressing the naturally occurring cis-4-proline hydroxylase showedincreased expression relative to the polynucleotide of SEQ ID NO:1. Theresidue differences from the first round screening were combined invarious permutations and screened for improved properties under HTPAssay, SFP Assay, and DSP Assay conditions. The engineered prolinehydroxylase polypeptide sequences and specific mutations and relativeactivities obtained from the screens are listed in Table 2A, 2B, 2C, 2D,2E, 2F, 2G, and 2H.

Example 2: Production of Engineered Proline Hydroxylases

The engineered proline hydroxylase polypeptides were produced in E. coliW3110 under the control of the lac promoter. Enzyme preparations forHTP, DSP, and SFP assays were made as follows.

High-Throughput (HTP) Growth, Expression, and Lysate Preparation.

Cells were picked and grown overnight in LB media containing 1% glucoseand 30 μg/mL chloramphenicol (CAM), 30° C., 200 rpm, 85% humidity. A 20μL aliquot of overnight growth was transferred to a deep well platecontaining 380 μL 2×TB growth media containing 30 μg/mL CAM, 1 mM IPTG,and incubated for ˜18 h at 30° C., 200 rpm, 85% humidity. Cell cultureswere centrifuged at 4000 rpm, 4° C. for 10 mM., and the media discarded.Cell pellets were resuspended in 100 μL Lysis Buffer (50 mM phosphatebuffer, pH 6.3, containing 100 μM Mohr's salt (i.e., (NH₄)₂Fe(SO₄)₂),0.5 mg/mL PMBS (polymyxin B sulfate) and 1 mg/mL Lysozyme). Lysis Bufferwas prepared fresh by adding to 60 mL of 50 mM phosphate buffer, pH 6.3,60 mg Lysozyme and 30 mg of PMBS. After mixing the Lysozyme solution,0.6 mL of 10 mM Mohr's salt solution (in H₂O) was added.

Production of Shake Flask Powders (SFP):

A shake-flask procedure was used to generate engineered prolinehydroxylase polypeptide powders used in secondary screening assays or inthe biocatalytic processes disclosed herein. Shake flask powder (SFP)provides a more purified preparation (e.g., up to 30% of total protein)of the engineered enzyme as compared to the cell lysate used in HTPassays. A single colony of E. coli containing a plasmid encoding anengineered polypeptide of interest is inoculated into 50 mL LuriaBertani broth containing 30 μg/ml chloramphenicol and 1% glucose. Cellsare grown overnight (at least 16 hours) in an incubator at 30° C. withshaking at 250 rpm. The culture is diluted into 250 mL Terrific Broth(12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mMpotassium phosphate, pH 7, 1 mM MgSO₄) containing 30 ng/mlchloramphenicol, in a 1 liter flask to an optical density of 600 nm(OD600) of 0.2 and allowed to grow at 30° C. Expression of the prolinehydroxylase gene is induced by addition of isopropyl-β-D-thiogalactoside(“IPTG”) to a final concentration of 1 mM when the OD600 of the cultureis 0.6 to 0.8. Incubation is then continued overnight (at least 16hours). Cells are harvested by centrifugation (5000 rpm, 15 mM, 4° C.)and the supernatant discarded. The cell pellet is resuspended with anequal volume of cold (4° C.) 50 mM potassium phosphate buffer, pH 6.3,and harvested by centrifugation as above. The washed cells areresuspended in two volumes of the cold 50 mM potassium phosphate buffer,pH 6.3 and passed through a French Press twice at 12,000 psi whilemaintained at 4° C. Cell debris is removed by centrifugation (9000 rpm,45 minutes, 4° C.). The clear lysate supernatant is collected and storedat −20° C. Lyophilization of frozen clear lysate provides a dryshake-flask powder of crude engineered polypeptide. Alternatively, thecell pellet (before or after washing) can be stored at 4° C. or −80° C.

Production of Downstream Process Powders (DSP):

DSP powders provide a more purified preparation of the engineeredproline hydroxylase enzyme as compared to the cell lysate used in theHTP or SFP assays. Larger-scale fermentation of the engineered prolinehydroxylase for production of DSP powders (˜100-120 g from 10L) can becarried out as a short batch followed by a fed batch process accordingto standard bioprocess methods. Briefly, proline hydroxylase expressionis induced by addition of IPTG to a final concentration of 1 mM.Following fermentation, the cells are harvested and resuspended in 50 mMphosphate buffer, then mechanically disrupted by homogenization. Thecell debris and nucleic acid are flocculated with polyethylenimine (PEI)and the suspension clarified by centrifugation. The resulting clearsupernatant is concentrated using a tangential cross-flowultrafiltration membrane to remove salts and water. The concentrated andpartially purified enzyme concentrate can then be dried in a lyophilizerand packaged (e.g., in polyethylene containers).

Example 3: Analytical Procedures

Method 1—HPLC Analysis of HTP Assay Reactions:

In a 96 deep well format assay block, 10 μL of reaction solution wasdiluted with 230 μL of 5% sodium bicarbonate solution followed by 160 μLof dansyl chloride solution (6 mg/mL dansyl chloride in MeCN). The platewas heat sealed, centrifuged, and placed in an incubator at 55° C. for45 minutes. The reaction solution turns from yellow to light yellow whenderivatization with dansyl chloride is complete. In cases where thesolution remained yellow, the plate was heated for another 15 mM. Afterincubation, the plate was centrifuged for 5 min at 4000 rpm. A 200 μLaliquot of supernatant was transferred into a 96 Corning plate for HPLCanalysis. The final concentration of the substrate was below 0.25 g/L.

The quenched reaction was subject to HPLC analysis under the followingconditions.

Column Agilent Poroshell120 SB-C18 50 × 4.6 mm (2.7 11 m) with guardcolumn Temperature 30° C. Mobile Phase Solution A: 2 mM Ammonium AcetatepH 3.3 Solution B: Acetonitrile Mobile Phase Profile Time: Flow rate:min A % B % mL/min 0.00 80 20 2.0 2.00 30 70 2.0 3.00 30 70 2.0 Postime1 min Detection Wavelength 250 nm Column Temperature 25° C. InjectionVolume 10 μL Total Runtime 4 min Response Factor N.A. (Substrate area/Product area)

Conversion of compound (2) to compound (1) was determined from theresulting chromatograms as follows:% Conversion={(RF×Product Area)/[(RF×Product Area)+Substrate Area]}×100whereResponse Factor (RF)=Substrate Area/Product Area.

This method was used for rapid identification for conversion of(2S)-piperidine-2-carboxylic acid to hydroxypiperidine-2-carboxylicacid.

The chromatographic elution profiles, denoted as “Response time”, are asfollows:

(2S,5S)-1-(5- (dimethylamino) naphthalene-1- ylsulfonyl)-5-hydroxypiperidine- 2-carboxylic acid

Response time: 1.59 min (2S,3R)-1-(5- (dimethylamino) naphthalene-1-ylsulfonyl)-3- hydroxypiperidine- 2-carboxylic acid

Response time: 1.76 min (5)-1-(5- (dimethylamino) naphthalene-1-ylsulfonyl) piperidine-2- carboxylic acid

Response time: 2.23 min

Method 2—HPLC Analysis of DSP and SFP Reactions:

A 10 μL of reaction solution from a DSP or SFP reaction was pipettedinto a 1.5 ml Eppendorf tube and diluted with 230 μL of 5% sodiumbicarbonate. A 160 μL aliquot of dansyl chloride solution (6 mg/mldansyl chloride in MeCN) was then added. The tube was heated with opencover at 55° C. for at least 30 minutes in a heating block to ensurefull derivatization, as indicated by change in color of thederivatization mixture from a yellow to a light yellow color. The tubewas vortexed and then centrifuged at 12,000 rpm for 5 minutes. A 200 μLaliquot of supernatant was transferred into a 2 ml HPLC vial withinsert. The vial was submitted to reverse phase HPLC-UV for analysis, asdescribed below. The final concentration of the substrate was below 0.25g/1.

The quenched reaction was subject to HPLC analysis under the followingconditions.

Column Supelco Ascentis Express C18 100 × 4.6 mm (2.7 11 m), attachedMobile Phase Solution A: 2 mM Ammonium Acetate, pH 3.3 Solution B:Acetonitrile Mobile Phase Profile Time: Flow rate: min A % B % mL/min 080 20 1.0 9.0 27.5 72.5 1.0 9.1 0 100 1.0 12.0 0 100 1.0 12.1 80 20 1.0Postime 2.90 min Detection Wavelength 250 nm Column Temperature 25° C.Injection Volume 10 μL Total Runtime 15.0 min Response Factor (RF) N.A.(Substrate area/ Product area)

The chromatographic elution profiles are as follows:

(2S,5R)-1-(5- (dimethylamino) naphthalene-1- ylsulfonyl)-5-hydroxypiperidine- 2-carboxylic acid

Response time: 5.16 min (2S,5S)-1-(5- (dimethylamino) naphthalene-1-ylsulfonyl)-5- hydroxypiperidine- 2-carboxylic acid

Response time: 5.57 min (2S,3R)-1-(5- (dimethylamino) naphthalene-1-ylsulfonyl)-3- hydroxypiperidine- 2-carboxylic acid

Response time: 6.02 min (5)-1-(5- (dimethylamino) naphthalene-1-ylsulfonyl) piperidine-2- carboxylic acid

Response time: 8.17 min

Example 4: High Throughput (HTP) Screening of Proline Hydroxylases forConversion of Compound (2) to Compound (1)

HTP Screening Assays: High-throughput screening used to guide primaryselection of variants was carried out in 96-well plates using celllysates. Two conditions, Condition A and Condition B, were used.

Condition A reactions were carried out as follows. Cells were grown in96-well plates as described above and lysates prepared by dispensing 100μL Lysis Buffer into each well. Lysis buffer was prepared by dissolving30 mg of lysozyme and 15 mg of PMBS in 30 mL of 50 mM phosphate buffer,pH 6.3. A 600 μL volume of 10 mM Mohr's salt, freshly prepared insterile water, was added to the lysozyme solution. The plate was heatsealed and then shaken on a titre-plate shaker at Speed #8 for 2 h atroom temperature. Subsequently, the plate was quick-spun to settle thelysate at the bottom of the plate. This crude lysate was to be used forthe reaction.

The Condition A reactions in 200 μL scale were carried out in 98 wellplates. A premix stock solution was prepared by dissolving 1.33 g ofα-ketoglutaric acid and 1.47 g of L-ascorbic acid in 31.5 mL of 50 mMphosphate buffer, pH 6.3 (pH adjusted by using KOH). After mixing, thepH was adjusted to 6.3 with KOH. To the pH adjusted premix, 41.16 mg ofMohr's salt was added. The solution turns cloudy due to the lowsolubility of Mohr's salt in aqueous solvents.

To each 100 μL of crude lysates prepared above, 90 μL of the premixstock solution was added into each well, followed immediately by 10uL/well of a 200 g/L substrate stock solution, i.e., compound (2)prepared in 50 mM phosphate buffer, pH 6.3. The plate was sealed with anAirPore seal (Qiagen) and the reaction left to proceed overnight on atitre-shaker, with shaking speed of #2.5 at room temperature.

Condition A has the following final reaction parameters: (a) 10 g/Lsubstrate loading; (b) 19 g/L α-ketoglutaric acid; (c) 21 g/L ascorbicacid; (d) 1.5 mM Mohr's salt; (e) 50 mM phosphate buffer, pH 6.3 (pHadjusted with KOH); (f) ambient temperature (20° C. to 25° C.); and (g)reaction time of about c.a. 24 h.

Following the overnight incubation, the plates were centrifuged at 4000rpm for 5 mM at room temperature. The reaction samples were derivatizedand quenched by aliquoting 10 μL of the clear reaction mix into a 96deep well plate containing 2304/well of 5% sodium bicarbonate (aq). A160 μL volume of 6 mg/mL of dansyl chloride in MeCN was added to eachwell, the plate heat sealed, and then quick spun to settle the reactionsolution to the bottom of the well. The plate was then heated at 55° C.for at least 45 mM without shaking, and centrifuged at 4000 rpm for 10min at room temperature. A 200 μL volume of the derivatized solution wastransferred into a 96 round bottom plate and submitted for HPLCanalysis.

Condition B reactions were carried out as follows. Cells were grown in96-well plates as described above and lysates prepared by dispensing 100μL of Lysis Buffer into each well. Lysis buffer was freshly prepared bydissolving 30 mg of lysozyme and 15 mg of PMBS in 30 mL of 50 mMphosphate buffer, pH 6.3, followed by 600 μL volume of 10 mM Mohr'ssalt, freshly prepared in sterile water. The lysis plate was heat sealedand then shaken on a titre-plate shaker at Speed #8 for 2 h at roomtemperature. Subsequently, the plate was quick-spun to settle the lysateat the bottom of the plate. This 100 μL crude lysate was to be used forthe reaction.

The Condition B reactions in 200 μL scale were carried out in 98 wellplates. A premix stock solution was prepared by dissolving 1.33 g ofα-ketoglutaric acid and 1.47 g of L-ascorbic acid in 31.5 mL of 50 mMphosphate buffer, pH 6.3 (pH adjusted by using KOH). After mixing, thepH was adjusted to 6.3 with KOH. To the pH adjusted premix, 41.16 mg ofMohr's salt was added.

To each 100 μL of crude lysates prepared above, 90 μL of the premixstock solution was added into each well, followed immediately by 10uL/well of a 200 g/L substrate stock solution, i.e., compound (2)prepared in 50 mM phosphate buffer, pH 6.3. The plate was sealed with anAirPore® seal (Qiagen) and the reaction left to proceed overnight on atitre-shaker, with shaking speed of #2.5 at room temperature.

Condition B has the following final reaction parameters: (a) 10 g/Lsubstrate loading; (b) 19 g/L α-ketoglutaric acid; (c) 21 g/L ascorbicacid; (d) 1.5 mM Mohr's salt; (e) 50 mM phosphate buffer pH 6.3 (pHadjusted with KOH); (f) ambient temperature (20° C. to 25° C.); and (g)reaction time of c.a. 24 h.

Following the overnight incubation, the plates were centrifuged at 4000rpm for 5 mM at room temperature. The reaction samples were derivatizedand quenched by aliquoting 10 μL of the clear reaction mix into a 96deep well plate containing 230 μL/well of 5% sodium bicarbonate (aq). A160 μL volume of 6 mg/mL of dansyl chloride in MeCN was added to eachwell, the plate heat sealed, and then quick spun to settle the reactionsolution to the bottom of the well. The plate was then heated at 55° C.for at least 45 mM with shaking on an Infors HT Microtron with a shakingspeed of 500 rpm. The plates were centrifuged at 4000 rpm for 10 mM atroom temperature. A 200 μL volume of the derivatized solution wastransferred into a 96 round bottom plate and submitted for HPLC foranalysis.

Example 5: Process for Conversion of Compound (2) to Compound (1) UsingShake Flask Powder (SFP) Preparations

A 200 μL scale reaction using SFP enzyme powder was carried out asfollows. A premix stock solution was freshly prepared by dissolving 1.05g of α-ketoglutaric acid, 420 mg of L-ascorbic acid, and 600 mg ofsubstrate (2S)-piperidine-2-carboxylic acid in 10 mL of 50 mM phosphatebuffer, pH 6.3 (pH adjusted by using KOH). After thoroughly mixing thesolution, the pH was adjusted to 6.3 using KOH. To the pH adjustedpremix solution, 45 mg of Mohr's salt was added.

A stock solution of enzyme was prepared by dissolving 20 mg of SFPenzyme powder into 2 mL of 50 mM phosphate buffer, pH 6.3. To initiatethe reaction, 100 μL of enzyme solution was added into a plate followedby 100 μL of premix stock solution for a final reaction volume of 200μL. The plate was sealed with an AirPore® seal (Qiagen) and the reactionallowed to proceed overnight (c.a., 24 h) with shaking on a titre-plateshaker (speed #2.5) at room temperature.

The SFP Assay condition (i.e., Condition C) has the following finalparameters: (a) 5 g/L enzyme powder loading; (b) 30 g/L substrateloading; (c) 52.5 g/L α-ketoglutaric acid; (d) 21 g/L L-ascorbic acid;(e) 2.25 mM Mohr's salt; (f) 50 mM potassium phosphate buffer, pH 6.3(pH adjusted with KOH), (g) reaction temperature at ambient roomtemperature; and (h) reaction time of about c.a. 24 h. In somereactions, the reaction conditions further contained 1% (v/v) Y-30®antifoam (Dow Corning), and the reaction solution was sparged with 02gas at 2 L/h.

The reactions were quenched with 400 μL of 75% MeCN and 25% H₂O. Theplates were shaken for 10 mM at room temperature and centrifuged at 4000rpm. Derivatization was carried out by transferring 20 μL of quenchedreaction to a 96 deep well plate containing 230 μL/well of 5% sodiumbicarbonate (aq). A 1504 aliquot of 21 mg/mL dansyl chloride in MeCN wasadded to each well. The plate was heat sealed and quick spun, and theplates incubated at 65° C. for at least 1 h with shaking on an Infors HTMicrotron at a shaking speed of 500 rpm. The plates were thencentrifuged at 4000 rpm for 10 mM at room temperature. A 200 μL volumeof the derivatized solution was transferred into a 96 round bottom plateand submitted for analysis by HPLC.

Example 6: Process for Conversion of Compound (2) to Compound (1) UsingDownstream Process Powder (DSP) Preparations

Two reaction conditions were used for downstream process powder (DSP)preparations. The first reaction conditions, referred to as “mini-DSP”conditions (i.e., Condition D) were carried out on a 1 mL scale asfollows. A premix stock solution was freshly prepared by dissolving 120mg of (2S)-piperidine-2-carboxylic acid (i.e., L-pipecolic acid), 228 mgof α-ketoglutaric acid and 252 mg of L-ascorbic acid in 11.88 mL of 50mM phosphate buffer, pH 6.3. The pH of the premix solution was thenadjusted to 6.3 using KOH. A 120 μL volume of 150 mM Mohr's salt (insterile water) was added to form the premix stock solution.

Reactions were run by weighing 20 mg of the DSP enzyme powder into avial followed by 1 mL of premix stock solution. The solution wasthoroughly mixed, and the vial left open overnight (˜24 h) at roomtemperature. The reaction solution was stirred at 1200 rpm during thecourse of the reaction.

The “mini DSP” reaction conditions have the following parameters: (a) 20g/L substrate loading; (b) 34 g/L α-ketoglutaric acid (1.5 equivalentsof substrate); (c) 13.6 g/L ascorbic acid (0.5 equivalents ofsubstrate); (d) 1.5 mM Mohr's salt; (e) 20 g/L protein of DSP enzymepreparation; (f) 50 mM phosphate buffer, pH 6.3 (pH adjusted with KOH);(f) ambient temperature; and (g) reaction time of ˜24 h. In somereactions, the reaction solution further contained 1% (v/v) of Y-30®antifoam (Dow Corning) and the reaction solution was sparged with O₂ gasat 2 L/h during the course of the reaction.

To follow the course of the reaction, 10 μL samples were taken anddissolved in 230 μL of 5% sodium bicarbonate (aqueous). A 160 μL volumeof 6 mg/mL of dansyl chloride in MeCN was then added to the mixture, thetubes thoroughly mixed, and then heated, uncapped at 50° C. for 30minutes. The samples were then centrifuged, and the clear supernatantanalyzed by HPLC as described in Example 2.

The second reaction conditions, referred to as “full DSP” conditions,were carried out as follows. A premix stock solution was freshlyprepared for 1 mL scale reactions by dissolving 240 mg of(2S)-piperidine-2-carboxylic acid (L-pipecolic acid), 228 mg ofα-ketoglutaric acid, and 252 mg of L-ascorbic acid in 11.88 mL of 50 mMphosphate buffer, pH 6.3. The pH of the premix solution was adjusted to6.3 with KOH. A 120 μL volume of 150 mM Mohr's salt (in sterile water)was added to form the premix stock solution.

Reactions were run by weighing 10 mg of the DSP enzyme powder and adding1 mL of premix stock solution. After mixing, the vial was left openovernight (˜24 h) at room temperature. The reaction solution was stirredat 1200 rpm during the course of the reaction.

The “full DSP” reaction conditions has the following parameters; (a) 10g/L substrate loading; (b) 38 g/L α-ketoglutaric acid; (c) 21 g/Lascorbic acid; (d) 1.5 mM Mohr's salt; (e) 10 g/L DSP enzymepreparation; (f) 50 mM phosphate buffer, pH 6.3 (pH adjusted with KOH);(f) reaction temperature of 25° C.; and (g) reaction time of c.a. 24 h.In some reactions, the reaction solution contained 1% (v/v) Y-30®antifoam (Dow Corning), and the reaction solution was sparged with 02gas at 2 L/h.

To follow the course of the reaction, 10 μL samples were removed andmixed with 230 μL of 5% sodium bicarbonate (aq). A 160 μL volume of 6mg/mL of dansyl chloride in MeCN was then added to the mixture. Thetubes were thoroughly mixed and then heated, uncapped at 50° C. for 30minutes. The samples were then centrifuged and the clear supernatantanalyzed by HPLC as described in Example 2.

Example 7: Process for Conversion of Compounds of Formula (II) toCompounds of Formula (I) Using DSP Powders of Engineered ProlineHydroxylase Polypeptides

The ability of the engineered proline hydroxylases to recognizesubstrates other than proline or pipecolic acid were examined. Thereaction conditions comprised (a) 20 g/L substrate loading; (b) 35 g/Lα-ketoglutaric acid; (c) 14 g/L ascorbic acid; (d) 1.5 mM Mohr's salt;(e) 10 g/L protein of DSP enzyme preparation of SEQ ID NO:108; (f) 50 mMphosphate buffer, pH 6.3 (pH adjusted with KOH); (f) reactiontemperature of 25° C.; and (g) reaction time of ˜24 h. The negativecontrols used enzyme preparations obtained from cells transformed withexpression vector that did not have a gene encoding a prolinehydroxylase.

Neg Substrate Substrate Structure Control Rxn Product(s) ProductStructure(s) L-pipecolic acid

— +++++ 2 regioisomers

L-proline

— ++++ 1 isomer

L-norvaline

+ ++ 2 isomers

S-(1,2,3,4)- tetrahydro-3- isoquinoline carboxylic acid

+ ++ 3 major products

The reactions were quenched by diluting 2000-fold in 50:50acetonitrile:H₂O, and the reaction products analyzed by LC/MS/MS.

LC/MS/MS analysis for pipecolic acid, proline and norvaline was carriedout under the following conditions:

Column ChiroBiotic TAG 250 × 4.6 mm, 5 μm Mobile Phase Solution A: 0.1%formic acid Solution B: 0.1% formic acid in Acetonitrile A:B = 50:50Postime 5.0 min MS conditions Source dependent parameters: CUR 30, IS5500, TEM 590° C., GS1 60, GS2 60, DP30, EP10, CE 20 MRM: 130/84(pipecolic acid RT 4.2 min), 146/100 and 146/82 (hydroxylated pipecolicacid 3.2 min and 3.7 min) MRM: 116/70 (proline RT 3.4 min), 132/86 and132/68 (hydroxylproline RT 2.7 min) MRM: 118/72 (norvaline RT 2.7 min),134/88, 134/74 and 134/70 (hydroxylated norvaline RT 2.6&2.7 min) ColumnNot controlled Temperature Injection 2 μL Volume

The quenched reaction for tetrahydroisoquinoline carboxylic acid wassubject to LC/MS/MS analysis under the following conditions:

Column Poroshell EC C18 100 × 4.6 mm, 2.7 μm Mobile Phase Solution A:0.5 mM perfluoroheptanoic acid Solution B: Acetonitrile Time Flow (min)(ml/min) A B 0-1.5 0.8 97 3  9 0.8 70 30 12 0.8 70 30 13 0.8 97 3 20 0.897 3 Postime 20 min MS conditions Source dependent parameters: CUR 30,IS 5500, TEM 600° C., GS1 60, GS2 60, DP30, EP10, CE 30 MRM: 194/148 and194/146 (hydroxylated tetrahydroisoquinoline carboxylic acid RT 5.7 min,7.26 min and 9.73 min) Column Not controlled Temperature Injection 2 μLVolume

Example 8: Process for Conversion of L-Pipecolic Acid (Compound (2)) to(2S,5S)-5-Hydroxypiperidine-2-Carboxylic Acid (Compound (1)) Followed byBoc-Protection Step

Enzymatic Reaction:

A solution of L-pipecolic acid (15 g) dissolved in 138 ml of 50 mMpotassium phosphate buffer, pH 6.3 was charged to a pre-mixed solutioncontaining: (i) DSP preparation of the polypeptide of SEQ ID NO: 132 (5g); (ii) Antifoam Y-30 emulsion (5 mL); (iii) Mohr's salt (1.08 g); (iv)α-ketoglutaric acid (25.5 g); and (iv) ascorbic acid (10.2 g); alldissolved in 250 mL of 50 mM potassium phosphate buffer, pH 6.3. Theresulting mixture was stirred and sparged with oxygen at a rate of 3 L/hand 25° C. The progress of the enzymatic reaction was monitored by HPLCusing Method 2 of Example 3. The conversion of L-pipecolic acid to(2S,5S)-5-hydroxypiperidine-2-carboxylic acid followed a reaction courseof ˜78% conversion at 25 h, ˜92% conversion at 45 h, and ˜94% conversionby 52 h, but did not reach higher conversion at 75h. The region purityof the (2S,5S)-5-hydroxypiperidine-2-carboxylic acid product after 52 hreaction was 6:1.

Boc-Protection:

Crude mixture from the enzymatic reaction was adjusted to pH 9.5 withKOH (50% w/w), heated to 60° C. for 1 h, and then cooled to roomtemperature. Thereafter, Celite filter-aid (15 g) was added withstirring (10 minutes) and the mixture was filtered through a 1 cm thickpad of Celite 545. The filter cake was washed with water (60 mL) and thefiltrate charged with NaOH (38.5 mL at 10 M) and di-tert-butyldicarbonate (Boc₂-O) (42.2 g in 75 mL THF). Upon reaction completion(˜96% conversion in two days) the aqueous phase was washed twice withheptanes (2×125 mL). The heptane washings were discarded and the aqueousphase was adjusted to pH 3.5 with 5 M HCl, and treated with NaCl (40 g).The aqueous phase was extracted with t-butyl methyl ether (TBME) (3×125mL), and the resulting organic phase extracts were combined, dried overMgSO₄, filtered and concentrated to give crude(2S,5S)-1-(tert-butoxycarbonyl)-5-hydroxypiperidine-2-carboxylic acid.

Isolation:

A solution of the crude(2S,5S)-1-(tert-butyloxycarbonyl)-5-hydroxypyridine-2-carboxylic acidproduct (25 g) in TBME (250 mL) was agitated with a magnetic stirrerbar, insolubles were filtered, and rinsed with TBME (150 mL). Theconcentrated filtrate was dried under vacuum overnight and dissolved inisopropyl acetate (100 mL) and heptane (100 mL). The resulting mixturewas heated at 80° C. for 20-25 minutes, insolubles were removed by hotfiltration, filtrate was cooled and stirred for 24 h at roomtemperature. The solid was filtered and the cake washed with chilled (0to 5° C.) heptanes-isopropyl acetate mixture (1:1 of 50 mL). Solidproduct was collected and dried under vacuum overnight to affordpurified(2S,5S)-1-(tert-butyloxycarbonyl)-5-hydroxypyridine-2-carboxylic acid(8.6 g, 30% yield).

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An engineered polypeptide having prolinehydroxylase activity, comprising an amino acid sequence having at least95% sequence identity to SEQ ID NO:2 and, wherein the engineeredpolypeptide has a residue difference at position X3 of SEQ ID NO:2. 2.The engineered polypeptide of claim 1, wherein the residue difference atposition X3 is X3S.
 3. The engineered polypeptide of claim 1, whereinthe amino acid sequence comprises one or more combination of featuresselected from the group consisting of: (a) X103L and X166Q; (b) X52P andX255Y; (c) X4E/L/S and X115A; (d) X25R and X58A; (e) X29A and X166T/Q/L;(f) X115H/D/G and X121F; (h) X103L, X131Y/F, and X166T/Q/L; (i) X26T,X103L and X166T/Q/L; (j) X25R, X66Q, X92V and X115E; and (k) X25R, X66Q,X92V, X103L, X115E, and X166Q.
 4. The engineered polypeptide of claim 1,further comprising one or more residue differences as compared to thesequence of SEQ ID NO: 2 at residue positions selected from: X17, X24,X26, X62, X88, X98, X114, X140, X151, X186, X188, and X205.
 5. Theengineered polypeptide of claim 4, wherein the residue differences atresidue positions X17, X24, X26, X62, X88, X98, X114, X140, X151, X186,X188, and X205 are selected from X17V, X24R, X24S, X26R, X26W, X62Q,X88R, X98F, X98T, X114N, X140L, X151A, X151H, X186G, X188G, and X205V.6. The engineered polypeptide of claim 1, wherein the polypeptideconverts substrate compound (2), (2S)-piperidine-2-carboxylic acid,

to product compound (1), (2S,5S)-5-hydroxypiperidine-2-carboxylic acid,

under suitable reaction conditions.
 7. The engineered polypeptide ofclaim 6, the polypeptide converts substrate compound (2) to productcompound (1) with at least 2 fold the activity of SEQ ID NO:2, andwherein the amino acid sequence comprises one or more residuedifferences selected from the group consisting of: X4Q; X4L; X5I; X5L;X24S; X25R; X30P; X66Q; X86S; X92V; X103L; X103Q; X113E; X115E; X150S;X166Q; X151S; X225L; and X270E.
 8. The engineered polypeptide of claim1, wherein the polypeptide converts substrate compound (2)

to product compound (1)

in excess of product compound (1a),(2S,3R)-3-hydroxypiperidine-2-carboxylic acid,

wherein the amino acid sequence comprises one or more residuedifferences selected from the group consisting of: X103L; X115E; X131Yand X166Q.
 9. The engineered polypeptide of claim 1, wherein thepolypeptide forms product compound (1),

in diastereomeric excess of product compound (1R),(2S,5R)-5-hydroxypiperidine-2-carboxylic acid,